A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells.

While human pluripotent stem cells are attractive sources for cell-replacement therapies, a major concern remains regarding their tumorigenic potential. Thus, safety assessment of human pluripotent stem cell-based products in terms of tumorigenicity is critical. Previously we have identified a pluripotent stem cell-specific lectin probe rBC2LCN recognizing hyperglycosylated podocalyxin as a cell surface ligand. Here we demonstrate that hyperglycosylated podocalyxin is secreted from human pluripotent stem cells into cell culture supernatants. We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN. The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells. This work provides a proof of concept for the noninvasive and quantitative detection of tumorigenic human pluripotent stem cells using cell culture supernatants. The developed method should increase the safety of human pluripotent stem cell-based cell therapies.

Identification of MIWI-associated Poly(A) RNAs by immunoprecipitation with an anti-MIWI monoclonal antibody.

MIWI is one of the PIWI subfamily of proteins mainly expressed in mouse germ cells, and associates with pachytene piRNAs. MIWI has been thought to play an essential role in spermatogenesis and spermiogenesis via biogenesis and/or stability of pachytene piRNAs, retrotransposon silencing, and post-transcriptional regulation of target mRNAs. However, MIWI's detailed role and function are not well understood. In this study, we produced an anti-MIWI mouse monoclonal antibody and identified MIWI-associated poly(A) RNAs by immunoprecipitation from adult mouse testes lysates. Approximately 70% of the MIWI-associated poly(A) RNAs were known mRNAs and 30% of them were unknown non-coding RNAs. These poly(A) RNAs contained piRNA-encoding RNAs transcribed from piRNA cluster regions and piRNA-encoding mRNA, such as Aym1 mRNA. Mature piRNAs specifically encoded in these piRNA-encoding RNAs were generated in pachytene spermatocytes and not detected in Miwi-deficient (Miwi-/-) testes. Moreover, MIWI associated with a large number of known mRNAs whose expression levels were increased in pachytene spermatocytes, and the expression of these mRNAs was decreased in Miwi-/- testes at 20 days postpartum when pachytene spermatocytes were most abundant. These results strongly suggest that MIWI is involved in pachytene piRNA biogenesis and the positive regulation of target mRNA metabolism in pachytene spermatocytes via association with pachytene piRNA precursors and target mRNAs.

Collagen type IV-specific tripeptides for selective adhesion of endothelial and smooth muscle cells.

Controlling the balance of endothelial cells (ECs) and smooth muscle cells (SMCs) in blood vessels is critically important to minimize the risk associated with vascular implants. Extracellular matrix (ECM) plays a key role in controlling the cellular balance, suggesting a promising source of cell-selective peptides. To obtain EC- or SMC-selective peptides, we start by highlighting sequence differences found among ECM molecules as enriched targets for cell-selective peptides. We explored the EC- or SMC-selective performance of tripeptides that are specifically enriched only in collagen type IV, but not in types I, II, III, and V. Collagen type IV was chosen since it is the major ECM in the basement membrane of blood vessels, which separates ECs and SMCs. Among 114 collagen type IV-derived tripeptides pre-screened from in silico analysis, 22 peptides (19%) were found to promote cell-selective adhesion, as determined by peptide array. One of the best performing EC-selective peptides (Cys-Ala-Gly (CAG)) was mixed into an electrospun fine-fiber, a vascular graft material, for practical application. Compared to unmodified fiber, the CAG containing fiber surface was found to enhance adhesion of ECs (+190%) while limiting SMCs (-20%). These results are not only consistent with the hypothesis of ECM as a source of cell selective peptides, but also suggest a new genre of EC- or SMC-selective peptides for tissue engineering applications. Collectively, these findings favorably support the screening approach used to discover new peptides for these purposes.

Coenzyme B12-dependent diol dehydratase is a potassium ion-requiring calcium metalloenzyme: evidence that the substrate-coordinated metal ion is calcium.

The X-ray analyses of coenzyme B(12)-dependent diol dehydratase revealed two kinds of electron densities that correspond to metal ions in the active site. One is directly coordinated by substrate [Shibata, N., et al. (1999) Structure 7, 997-1008] and the other located near the adenine ring of the coenzyme adenosyl group [Masuda, J., et al. (2000) Structure 8, 775-788]. Both have been assigned as potassium ions, although the coordination distances of the former are slightly shorter than expected. We examined the possibility that the enzyme is a metalloenzyme. Apodiol dehydratase was strongly inhibited by incubation with EDTA and EGTA in the absence of substrate. The metal analysis revealed that the enzyme contains approximately 2 mol of tightly bound calcium per mole of enzyme. The calcium-deprived, EDTA-free apoenzyme was obtained by the EDTA treatment, followed by ultrafiltration. The activity of the calcium-deprived apoenzyme was dependent on Ca(2+) when assayed with 1 mM substrate. The K(m) for Ca(2+) evaluated in reconstitution experiments was 0.88 muM. These results indicate that the calcium is essential for catalysis. Ca(2+) showed a significant stabilizing effect on the calcium-deprived apoenzyme as well. It was thus concluded that the substrate-coordinated metal ion is not potassium but calcium. The potassium ion bound near the adenine ring would be the essential one for the diol dehydratase catalysis. Therefore, this enzyme can be considered to be a metal-activated metalloenzyme.

Observation of interactions of human serum components with transferrin by affinity capillary electrophoresis.

Interaction of human transferrin (TF) with human serum components was investigated by affinity capillary electrophoresis. It was found that any peaks of human serum protein fractions did not give migration time change on addition of intact TF to running buffer (50mM phosphate buffer, pH 7.5), whereas two peaks belonging to alpha-globulin fraction showed marked acceleration upon addition of desialylated TF. These results provide strong evidence that the sialic acid residue in TF masks its binding ability to serum proteins. The association constants of desialylated TF to these interactive components, estimated based on the double reciprocal plot of migration time change vs. glycoprotein concentration, were at a high level of 10(7)M(-1). TF is well known as a ferric ion transfer protein, and hence formation of this protein might be changed by ferric ion. The presence of iron(II) played no essential role in this interaction, though its influence was not negligible.

Purification of boron nitride nanotubes through polymer wrapping.

An effective method was proposed to remove obstinate boron nitride phase impurities in boron nitride nanotubes (BNNTs). The method is based on strong interactions between BNNTs and a conjugated polymer wrapping them and significant weight and size difference between BNNTs and impurities. The as-grown samples and purified samples were compared through detailed characterization, using scanning electron microscopy, transmission electron microscopy, and Raman and Fourier transformed infrared spectroscopy. The results reveal that impurities are effectively removed and resultant BNNTs possess perfect crystallization.

A sulfonated capillary that gives reproducible migration times for capillary zone electrophoresis and micellar electrokinetic chromatography.

To obtain reproducible migration times and rapid analyses of analytes, sulfonate groups were chemically introduced to the inner wall of untreated fused-silica capillary with 2-(4-chlorosulfonylphenyl)ethyltrichlorosilane. The sulfonated capillary showed relatively constant electroosmotic mobility which was greater than that obtained by an untreated fused-silica capillary over the pH range studied (pH 2-9). In both CZE and MEKC, the RSDs of the migration times of analytes with the sulfonated capillary were less than 0.2% which were significantly lower than those obtained with an untreated fused-silica capillary (0.5-3.5%). When BGE were set at pH 7.0 for CZE and MEKC, the analysis times with the sulfonated capillary were about half those obtained with an untreated fused-silica capillary. These results indicate that the sulfonated capillary can provide highly reproducible and rapid analyses in CE.

Search for serum protein-binding disaccharides and disaccharide-binding serum proteins by affinity capillary electrophoresis.

The potential use of affinity capillary electrophoresis in a microscale search for mutually interacting substances in biological fluid is demonstrated. Some disaccharides, especially gentiobiose (Gen), derivatized with 1-phenyl-3-methyl-5-pyrazolone, caused peak retardation when electrophoresed in a neutral running buffer, containing human serum. Gen, the most significantly retarded disaccharide, was converted to its negatively charged bis-mercaptoethanesulfonate derivative (MerESGen), and a serum sample was analyzed in a neutral buffer containing the derivatized disaccharide. Two peaks, belonging to the beta-globulin fraction, were found to be remarkably retarded in the buffer containing MerES-Gen in a concentration-dependent way. These findings prove an interaction between disaccharides and serum proteins.

Miniaturization in carbohydrate analysis.

Recent progress of microchip electrophoresis (ME) of carbohydrates is overviewed. Carbohydrate analysis by ME encounters difficulties such as lack of electric charge and deficiency of a chromophore/fluorophore in analyte molecules, however, it benefits from the accumulated knowledge of capillary electrophoresis (CE) and rapid separation of simple sugars also by ME, with high column efficiency comparable to CE, has become possible. Analysis at high pH, with electrochemical detection, is a promising approach because carbohydrates can be ionized by weak dissociation of the hydroxyl groups and the in situ formed ionic species can be effectively separated by the zone electrophoresis mode. The separated species can be sensitively monitored by electrochemical detection on a gold or copper electrode. Ionization as borate complexes and refractometric detection is also possible, though sensitivity is lower. Introduction of UV-absorbing or fluorescent tags is potentially useful but the time-consuming derivatization processes sacrifice the rapidity of ME. Examples of ME of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone (PMP; for simple mono- and oligosaccharides with UV detection), 8-aminopyrene-1,3,6-trisulfonate (APTS; for oligosaccharides ladders with LIF detection), and 4-nitro-2,1,3-benzoxadiazole (NBD-F; for amino sugars and aminoalditols with LIF detection) derivatives are presented, with details of the analytical conditions. Since ME in a short separation channel enables rapid analysis within 1 min, it presents an ideal tool for clinical analysis, as shown in a few papers reporting protocols for specific blood glucose assay. Finally, the usefulness of microfluidic reactors and microarrays for enzyme-assisted carbohydrate analysis as well as glycan profiling is pointed out.

High-speed electrophoretic analysis of 1-phenyl-3-methyl-5-pyrazolone derivatives of monosaccharides on a quartz microchip with whole-channel UV detection.

1-Phenyl-3-methyl-5-pyrazolone (PMP) derivatives of monosaccharides were analyzed by electrophoresis on a quartz microchip with whole-channel UV detection. Rapid separation of PMP derivatives of aldopentoses was achieved by plain-zone electrophoresis in a neutral phosphate buffer with the height equivalent to a theoretical plate at the micrometer level. Zone electrophoresis as borate complexes was also successful for the separation of PMP derivatives of a few aldoses, which were separated within 1 min. Separation by microchip electrophoresis was compared to that by capillary electrophoresis, and the difference was discussed in terms of column efficiency and sample column capacity.

Capillary electrophoretic separation of drug enantiomers in human serum.

Enantiomers of various solutes including several basic drugs and alpha-amino acids were analyzed by capillary electrophoresis in diluted human serum, and chloroquine and tryptophan were found to be well enantioseparated. In order to specify the protein responsible for enantioseparation, these drug enantiomers were analyzed in the presence of various serum protein fractions. The results indicated that albumin fraction caused enantioseparation but the alpha and beta -globulin mixed fraction, the gamma-globulin fraction and the alpha(1)-acid glycoprotein fraction did not exhibit any enantioseparation. The association constants between these drugs and albumin were roughly estimated based on our method. Approximate values were 1.50 x 10(3) and 1.85 x 10(3) M(-1) for chloroquine enantiomers, and 1.51 x 10(4) and 2.45 x 10(4) M(-1) for tryptophan enantiomers. The difference of the association constant values between the enantiomers was found to be 19% for chloroquine and 38% for tryptophan, when calculated based on the slower moving enantiomers.