Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

Adiponectin concentrations: a genome-wide association study

Adiponectin is associated with obesity and insulin resistance. To date, there has been no genome-wide association study (GWAS) of adiponectin levels in Asians. Here we present a GWAS of a cohort of Korean volunteers. A total of 4,001 subjects were genotyped by using a genome-wide marker panel in a two-stage design (979 subjects initially and 3,022 in a second stage). Another 2,304 subjects were used for follow-up replication studies with selected markers. In the discovery phase, the top SNP associated with mean log adiponectin was rs3865188 in CDH13 on chromosome 16 (p = 1.69 × 10(-15) in the initial sample, p = 6.58 × 10(-39) in the second genome-wide sample, and p = 2.12 × 10(-32) in the replication sample). The meta-analysis p value for rs3865188 in all 6,305 individuals was 2.82 × 10(-83). The association of rs3865188 with high-molecular-weight adiponectin (p = 7.36 × 10(-58)) was even stronger in the third sample. A reporter assay that evaluated the effects of a CDH13 promoter SNP in complete linkage disequilibrium with rs3865188 revealed that the major allele increased expression 2.2-fold. This study clearly shows that genetic variants in CDH13 influence adiponectin levels in Korean adults.     

Iron chelator triggers inflammatory signals in human intestinal epithelial cells: involvement of p38 and extracellular signal-regulated kinase signaling pathways

Competition for cellular iron (Fe) is a vital component of the interaction between host and pathogen. Most bacteria have an obligate requirement for Fe to sustain infection, growth, and survival in host. To obtain iron required for growth, many bacteria secrete iron chelators (siderophores). This study was undertaken to test whether a bacterial siderophore, deferoxamine (DFO), could trigger inflammatory signals in human intestinal epithelial cells as a single stimulus. Incubation of human intestinal epithelial HT-29 cells with DFO increased the expression of IL-8 mRNA, as well as the release of IL-8 protein. The signal transduction study revealed that both p38 and extracellular signal-regulated kinase-1/2 were significantly activated in response to DFO. Accordingly, the selective inhibitors for both kinases, either alone or in combination, completely abolished DFO-induced IL-8 secretion, indicating an importance of mitogen-activated protein kinases pathway. These proinflammatory effects of DFO were, in large part, mediated by activation of Na(+)/H(+) exchangers, because selective blockade of Na(+)/H(+) exchangers prevented the DFO-induced IL-8 production. Interestingly, however, DFO neither induced NF-kappaB activation by itself nor affected IL-1beta- or TNF-alpha-mediated NF-kappaB activation, suggesting a NF-kappaB-independent mechanism in DFO-induced IL-8 production. Global gene expression profiling revealed that DFO significantly up-regulates inflammation-related genes including proinflammatory genes, and that many of those genes are down-modulated by the selective mitogen-activated protein kinase inhibitors. Collectively, these results demonstrate that, in addition to bacterial products or cell wall components, direct chelation of host Fe by infected bacteria may also contribute to the evocation of host inflammatory responses.     

Improved Intraoperative Visualization of Nerves through a Myelin-Binding Fluorophore and Dual-Mode Laparoscopic Imaging

The ability to visualize and spare nerves during surgery is critical for avoiding chronic morbidity, pain, and loss of function. Visualization of such critical anatomic structures is even more challenging during minimal access procedures because the small incisions limit visibility. In this study, we focus on improving imaging of nerves through the use of a new small molecule fluorophore, GE3126, used in conjunction with our dual-mode (color and fluorescence) laparoscopic imaging instrument. GE3126 has higher aqueous solubility, improved pharmacokinetics, and reduced non-specific adipose tissue fluorescence compared to previous myelin-binding fluorophores. Dosing and kinetics were initially optimized in mice. A non-clinical modified Irwin study in rats, performed to assess the potential of GE3126 to induce nervous system injuries, showed the absence of major adverse reactions. Real-time intraoperative imaging was performed in a porcine model. Compared to white light imaging, nerve visibility was enhanced under fluorescence guidance, especially for small diameter nerves obscured by fascia, blood vessels, or adipose tissue. In the porcine model, nerve visualization was observed rapidly, within 5 to 10 minutes post-intravenous injection and the nerve fluorescence signal was maintained for up to 80 minutes. The use of GE3126, coupled with practical implementation of an imaging instrument may be an important step forward in preventing nerve damage in the operating room.     

Caspase-dependent cytochrome c release and cell death in chick cardiomyocytes after simulated ischemia-reperfusion

We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 +/- 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH(2)F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.     

Single-nucleotide polymorphisms and haplotype LD analysis of the 29-kb IGF2 region on chromosome 11p15.5 in the Korean population

OBJECTIVE: We investigated sequence variations of the 29-kb insulin-like growth factor 2 (IGF2) region in human chromosome region 11p15.5 in the Korean population. This region consists of IGF2, insulin-like growth factor 2 antisense (IGF2AS), and the insulin gene, all important candidate genes for various diseases, including cancer, obesity, diabetes, and coronary disease. While single nucleotide polymorphisms (SNPs) have been identified for this region and used in association studies, ethnic differences in genetic variation at this site have not been addressed. To date, SNPs for the entire 29-kb region in the Korean population have not been reported. METHODS: We surveyed a population of 108 Koreans for SNPs in the 29-kb IGF2 region. RESULTS: We identified 62 SNPs, consisting of 6 SNPs in the promoter region, 17 in the untranslated region, 19 in introns, and 20 in the intergenic region. We also analyzed linkage disequilibrium (LD) patterns and haplotypes using 36 high-frequency (> 5%)SNPs and found a well-defined LD block spanning about 13 kb that includes 8 kb of the IGF2AS gene, with two hot-spot regions flanking the LD block. CONCLUSION: These SNPs may be useful as genetic markers in disease association studies in the Korean population.