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.

Estimation of sialic acid in a sialoglycan and a sialoglycoprotein by capillary electrophoresis with in-capillary sialidase digestion.

An example of application of in-capillary derivatization for CE, obtained by using the throughout-capillary format, is presented. Introduction of a sialoglycan (N-acetylneuraminyllactose) or a sialoglycoprotein (bovine serum fetuin) sample to a running buffer (pH 5.0) containing N-acetylneuraminidase followed by application of a voltage resulted in the release of N-acetylneuraminic acid (NANA) which could be estimated by CE with UV detection. Two-step application of voltages (5 and 20 kV) was proved to be more effective for rapid estimation of the released NANA. This format (modified throughout-capillary format) allowed differential estimation of the NANA present in the sample as an impurity and the NANA released from the substrate at the picomol level, and thereby reliable micro assay of the sialidase activity. It also allowed estimation of the rate constant of this enzymatic reaction.

Effect of structure modification of chondroitin sulfate C on its enantioselectivity to basic drugs in capillary electrophoresis.

The effect of structure modification of chondroitin sulfate C on its enantioselectivity to several representative basic drugs in capillary electrophoresis was investigated. Chemical desulfation showed no remarkable decrease in selectivity, whereas depolymerization with chondroitinase ABC resulted in complete loss of selectivity. Comparison with chondroitin sulfate A indicated considerable decrease in selectivity with this isomer. The great retention of enantioselectivity in the desulfated derivative suggests that the selectivity comes from the difference of the magnitude of an interaction in the multipoint mechanism between a part of the drug molecule and a functional group in chondroitin sulfate C other than the sulfate group. The sulfate group is not considered to play a major role for chiral separation. The complete loss of selectivity by depolymerization is consistent with a general tendency of lower selectivity in smaller saccharides, and the priority of chondroitin sulfate C to chondroitin sulfate A suggests the importance of the hydroxyl group at C4 in the galactosamine residue. During the course of this work we observed heavy tailing of the peaks of basic drugs in some batches of uncoated fused-silica capillaries under acidic conditions and solved this problem by doubly coating capillaries with Polybrene followed by chondroitin sulfate C. On the other hand, we demonstrated the usefulness of a special technique which uses a short, wider bore PTFE tube-attached capillary for the study of the effect of depolymerization, in order to minimize sample amount.

Colominic acid: a novel chiral selector for capillary electrophoresis of basic drugs.

We introduced colominic acid as a new chiral selector for capillary electrophoresis of basic drugs. Use of a low concentration phosphate buffer containing this polysaccharide and a Polybrene/colominic acid double coated capillary allowed excellent separation of the enantiomers of primaquine, chloroquine and tryptophan. Other drugs giving partial enantioseparation include laudanosine and salbutamol. Capillary coating with Polybrene followed by colominic acid eliminated the problems of peak tailing and low reproducibility of migration time in uncoated capillaries. The optimum pH was in the acidic region but varied among drugs. A low capillary temperature of 16 degrees C and a colominic acid concentration of 9 w/v% are recommended for practical analysis of these drugs. Colominic acid preparations having higher molecular masses gave better enantioseparation, and N-acetylneuraminic acid, the component monosaccharide, did not give any enantioseparation.

Analysis of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone derivatives by capillary/microchip electrophoresis and capillary electrochromatography.

The 1-phenyl-3-methyl-5-pyrazolone (PMP) method has many advantages over hitherto reported methods based on reductive amination and hydrazone formation. This short review summarizes the various aspects of the PMP method, including the principle of derivatization, the simplicity of derivatization procedure, the high sensitivities to UV monitoring and ESI-MS, and the diversity of separation modes in capillary electrophoresis, and presents a number of application data for carbohydrate analysis in biological samples by this method. It also describes successful automation of carbohydrate analysis by in-capillary derivatization with PMP and miniaturization to microchip electrophoresis with whole channel UV detection allowing rapid (within 1 min) analysis of small amounts of PMP derivatives of carbohydrates. Furthermore, it discusses the possibility of capillary electrochromatography in carbohydrate analysis as PMP derivatives, and proposes an in-capillary modification strategy for improving column efficiency and elution time reproducibility.

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.

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.