8 research outputs found
Elucidating the effect of different amino-functionalized spherical mesoporous silica characteristics on ribonucleic acid selectivity and adsorption capacity
<p>Purifying ribonucleic acid (RNA) obtained from cells is an essential process in gene analysis and is generally performed using a polythymine oligonucleotide column and an organic solvent. However, these procedures are expensive and complicated. In the present study, we discovered that amino silica with a particle size of 50 nm was able to selectively adsorb RNA and investigated how varying the parameters of this material (i.e. mesopore existence, particle size, surface amino content, and pore diameter) affected the amount of RNA it could adsorb and its selective adsorption ability. Particle size and surface amino content were found to be important factors for RNA/deoxyribonucleic acid (DNA) value (RNA adsorption amount/DNA adsorption amount), and the presence of mesopores was found to promote the amount of nucleic acid that could be adsorbed. Mesoporous silica (MPS) with a particle size of 50 nm was synthesized using a 12/1 ratio of tetraethoxysilne/aminopropyltriethoxysilane, which acts as a silica source. The MPS was also found to have a high RNA adsorption capacity (272.6 μg/mg) and RNA selective adsorption ability (RNA/DNA value = 11.1).</p
pH Switching That Crosses over the Isoelectric Point (pI) Can Improve the Entrapment of Proteins within Giant Liposomes by Enhancing Protein–Membrane Interaction
The ability to encapsulate various
molecules including proteins
within giant liposomes is needed for studies on model cell membranes
and artificial cells. In this report, we demonstrate how to improve
the efficiency of protein entrapment with the gentle hydration (natural
swelling) method, which is a well-known protocol for the preparation
of giant liposomes. We found that when the initial pH of a solution
was kept below the pI of a target protein during hydration and then
changed to above the pI, the protein could be entrapped more efficiently
compared to the sample that was kept at above the pI during the hydration.
An examination of the ratio of the mass of entrapped protein to the
moles of phospholipid in liposomes (dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylglycerol
(DOPG)) indicated that entrapment of target proteins like bovine serum
albumin, myoglobin, and lysozyme could be improved using this procedure,
and this trend was consistent with microscopic observations at the
level of a single giant liposome. The conditions that resulted in
good efficiencies were affected by the NaCl concentration and the
temperature of the hydration solution, implying that protein entrapment
in giant liposomes may be enhanced by associative interaction between
lipid lamellar membranes and target proteins
Cell Pairing Using Microwell Array Electrodes Based on Dielectrophoresis
We report a simple device with an
array of 10 000 (100 ×
100) microwells for producing vertical pairs of cells in individual
microwells with a rapid manipulation based on positive dielectrophoresis
(p-DEP). The areas encircled with micropoles which fabricated from
an electrical insulating photosensitive polymer were used as microwells.
The width (14 μm) and depth (25 μm) of the individual
microwells restricted the size to two vertically aligned cells. The
DEP device for the manipulation of cells consisted of a microfluidic
channel with an upper indium tin oxide (ITO) electrode and a lower
microwell array electrode fabricated on an ITO substrate. Mouse myeloma
cells stained in green were trapped within 1 s in the microwells by
p-DEP by applying an alternating current voltage between the upper
ITO and the lower microwell array electrode. The cells were retained
inside the wells even after switching off the voltage and washing
with a fluidic flow. Other myeloma cells stained in blue were then
trapped in the microwells occupied by the cells stained in green to
form the vertical cell pairing in the microwells. Cells stained in
different colors were paired within only 1 min and a pairing efficiency
of over 50% was achieved
Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities
<div><p>Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain <i>N</i>-glycans at the constant region of the heavy chain (Fc domain), and their <i>N</i>-glycosylation patterns differ during various processes or among cell expression systems. The Fc <i>N</i>-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc <i>N</i>-glycans, we performed a rearrangement of Fc <i>N</i>-glycans from a heterogeneous <i>N</i>-glycosylation pattern to homogeneous <i>N</i>-glycans using chemoenzymatic approaches with two types of endo-β-<i>N</i>-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous <i>N</i>-glycans, another that is used as a glycosynthase to generate homogeneous <i>N</i>-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man<sub>2-3</sub>GlcNAc<sub>2</sub>), high-mannose type (Man<sub>4-9</sub>GlcNAc<sub>2</sub>), and complex type (Man<sub>3</sub>GlcNAc<sub>3-4</sub>) <i>N</i>-glycans. As a result of the cleavage of several ENG’ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired <i>N</i>-glycans (M3; Man<sub>3</sub>GlcNAc<sub>1</sub>, G0; GlcNAc<sub>2</sub>Man<sub>3</sub>GlcNAc<sub>1</sub>, G2; Gal<sub>2</sub>GlcNAc<sub>2</sub>Man<sub>3</sub>GlcNAc<sub>1</sub>, A2; NeuAc<sub>2</sub>Gal<sub>2</sub>GlcNAc<sub>2</sub>Man<sub>3</sub>GlcNAc<sub>1</sub>) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous <i>N</i>-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.</p></div
ENG’ase activity of the anti-Her2 mAbs (a; endoS, b; endoD, c; endoH, d; endoM, e; endoLL).
<p>(Blue bar represents glycopeptides without ENG’ase hydrolysis; red bar represents the remaining glycopeptides with ENG’ase hydrolysis; y-axis indicates each individual glycoform ratio to total glycoform content; % represents total cleaved glycopeptide ratio by ENG’ase hydrolysis.)</p
Binding activity for FcγRIIIa of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) using the FcγRIIIa-V158-binding ELISA method.
<p>Binding activity for FcγRIIIa of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) using the FcγRIIIa-V158-binding ELISA method.</p
MALDI QIT-TOF MS spectrum of Bz-labeled glycopeptides from anti-Her2 mAbs produced in silkworm cocoon.
<p>MALDI QIT-TOF MS spectrum of Bz-labeled glycopeptides from anti-Her2 mAbs produced in silkworm cocoon.</p
ADCC reporter gene assay of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) in SKBR-3 (a) and BT474 (b) target cells.
<p>ADCC reporter gene assay of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) in SKBR-3 (a) and BT474 (b) target cells.</p