Two-component membrane lithography via lipid backfilling.
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Journal Article↗
Biomedical subjects
Seung-Yong Jung
Publications and source records attributed to Seung-Yong Jung.
Creating fluid and air-stable solid supported lipid bilayers.
Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.
Air↗
Patterning enzymes inside microfluidic channels via photoattachment chemistry.
We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.
Animals↗
Production of interleukin-8 by human neutrophils stimulated with Trichomonas vaginalis.
Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients infected with Trichomonas vaginalis. Although chemoattractants, such as leukotriene B(4) and interleukin-8 (IL-8), are found in the vaginal discharges of symptomatic trichomoniasis patients, little is known about the mechanism of how neutrophils accumulate or mediate initial inflammatory response after acute T. vaginalis infection. We examined IL-8 production in neutrophils activated by T. vaginalis and evaluated the factors involved in T. vaginalis adherence that might affect IL-8 production. When human neutrophils were stimulated with live trophozoites, T. vaginalis lysate, or T. vaginalis excretory-secretory products, the live trichomonads induced higher levels of IL-8 production than the lysate or products did. When live trichomonads were pretreated with various inhibitors of proteinase, microtubule, microfilament, or adhesin (which are all known to participate in the adherence of T. vaginalis to vaginal epithelial cells), IL-8 production significantly decreased compared with the untreated controls. Furthermore, an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), a mitogen-activated protein (MAP) kinase (MEK) inhibitor (PD98059), and a p38 MAP kinase inhibitor (SB203580) significantly suppressed IL-8 synthesis in neutrophils. These results suggest that live T. vaginalis, particularly adherent trophozoites, can induce IL-8 production in neutrophils and that this action may be mediated through the NF-kappaB and MAP kinase signaling pathways. In other words, T. vaginalis-induced neutrophil recruitment may be mediated via the IL-8 expressed by neutrophils in response to activation by live T. vaginalis.
Animals↗
The Vroman effect: a molecular level description of fibrinogen displacement.
The molecular level details of the displacement of surface adsorbed fibrinogen from silica substrates were studied by atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy. The results showed that human plasma fibrinogen (HPF) can be readily displaced from the interface by other plasma proteins near neutral pH because the positively charged alpha C domains on HPF sit between the rest of the macromolecule and the underlying surface. The alpha C domains make weak electrostatic contact with the substrate, which is manifest by a high degree of alignment of Lys and Arg residues. Upon cycling through acidic pH, however, the alpha C domains are irreversibly removed from this position and the rest of the macromolecule is free to engage in stronger hydrogen bonding, van der Waals, and hydrophobic interactions with the surface. This results in a 170-fold decrease in the rate at which HPF can be displaced from the interface by other proteins in human plasma.
Adsorption↗
Development and applications of a beta-catenin oligonucleotide microarray: beta-catenin mutations are dominantly found in the proximal colon cancers with microsatellite instability.
beta-catenin mutations have been identified in a variety of human malignancies; most of these are missense mutations restricted at hot-spot areas in exon 3. beta-catenin mutations are known to be highly associated with colorectal cancers with microsatellite instability (MSI). More than 70 beta-catenin mutations have been reported in colorectal cancers, and approximately 90% of beta-catenin mutations have been found in 11 codons (codons 29, 31, 32, 33, 34, 35, 37, 38, 41, 45, and 48) as missense mutations or in-frame deletions. We have developed an oligonucleotide microarray for detecting beta-catenin mutations at these 11 codons. The developed oligonucleotide microarray can detect a total of 110 types of beta-catenin mutations, including the 60 mutations reported previously. Nine beta-catenin mutations were identified in this study by five different methods, i.e., PCR- single-strand conformational polymorphism, denaturing high performance liquid chromatography, direct sequencing, cloning-sequencing, and with an oligonucleotide microarray. All nine of the mutations were identified by denaturing high performance liquid chromatography, cloning-sequencing, and by the oligonucleotide microarray. However, PCR-single-strand conformational polymorphism missed 1 beta-catenin mutation and direct sequencing missed 2. Five beta-catenin mutations from 74 colorectal carcinomas (34 proximal colon cancers and 40 distal colorectal cancers) and 4 beta-catenin mutations from 31 colorectal cancer cell lines (7 from the proximal colon, 6 from the distal colorectum, and 18 unknown) were identified. In colorectal carcinomas, all 5 of the beta-catenin mutations were found in proximal colon tumors. In colorectal cancer cell lines, 2 of 4 cell lines with beta-catenin mutations originated from the proximal colon, and the remaining 2 cell lines were simply described as having originated from the colon. Considering the relationships among beta-catenin mutations, MSI, and tumor location, the frequency of beta-catenin mutations was found to be meaningfully higher in colorectal carcinomas with MSI than in those with microsatellite stability (P < 0.001); moreover, MSI was found to be more frequent in proximal colon tumors (P < 0.01). In addition, beta-catenin mutations were also found to be associated with proximal colon cancer (P = 0.017).
Adult↗