Conformation study on heparin by intermediate-angle X-ray scattering

The conformation of heparin in water was investigated by intermediate-angle x-ray scattering (IAXS). The theoretical scattering function for the coil conformation was calculated by the Monte Carlo method using the approximation of separable conformation energies and the conformation energies computed for two disaccharide pairs in heparin. From x-ray scattering in a relatively small-angle region, the conformation of heparin is not the ordered 2₁ helix conformation but the coil conformation obtained by the Monte Carlo calculation. It is expected, from x-ray scattering in a relatively wide-angel region, that the sulfate groups of heparin maintain about 7 Å between them.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins II: public release of inaugural version of InGaP database containing gene/protein expression profiles for 127 mouse KIAA genes/proteins.

The inaugural version of the InGaP database (Integrative Gene and Protein expression database; http://www.kazusa.or.jp/ingap/index.html) is a comprehensive database of gene/protein expression profiles of 127 mKIAA genes/proteins related to hypothetical ones obtained in our ongoing cDNA project. Information about each gene/protein consists of cDNA microarray analysis, subcellular localization of the ectopically expressed gene, and experimental data using anti-mKIAA antibody such as Western blotting and immunohistochemical analyses. KIAA cDNAs and their mouse counterparts, mKIAA cDNAs, were mainly isolated from cDNA libraries derived from brain tissues, thus we expect our database to contribute to the field of neuroscience. In fact, cDNA microarray analysis revealed that nearly half of our gene collection is predominantly expressed in brain tissues. Immunohistochemical analysis of the mouse brain provides functional insight into the specific area and/or cell type of the brain. This database will be a resource for the neuroscience community by seamlessly integrating the genomic and proteomic information about the mouse KIAA genes/proteins.     

Rapid and simple gas chromatographic measurement of lactic acid in red blood cells, plasma, and tumor cells after hyperthermia.

The content of lactic acid in red blood cells, plasma, and Ehrlich ascites tumor cells were measured by a gas-liquid chromatography using a column with a terephtalic acid support coated with polyethylene glycol-6000. The lactic acid contents were directly determined in aqueous samples, because they were converted to a volatile derivative in the column. The method was rapid and simple, compared with previous methods which need time-consuming conversion of lactic acid to volatile derivatives. Our measurements showed the increase in the contents of intra- and extracellular lactic acid after hyperthermia.     

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.     

Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans

A systematic method for ¹³C labeling of the glycan of immunoglobulin G for NMR study has been developed. A mouse immunoglobulin of subclass IgG2b has been used for the experiment. On the basis of chemical shift and linewidth data, it has been concluded that (1) the mobility of the carbohydrate chain in IgG2b is comparable to that of the backbone polypeptide chain with the exception of the galactose residue at the nonreducing end of the Man1–3 branch, which is extremely mobile and (2) agalactosylation does not induce any significant change in the mobility. The results obtained indicate that even in the agalactosyl form the glycans are buried in the protein. Biological significance of the NMR results obtained is also briefly discussed.     

Direct Repeat 3-Type Element Lacking the Ability to Bind to the Vitamin D Receptor Enhances the Function of a Vitamin D-responsive Element

In a previous study, we identified the element which allows the maximum response to 1,25(OH)₂D₃ in concert with two vitamin D-responsive elements (VDREs) in the rat 25-hydroxyvitamin D₃ 24-hydroxylase gene promoter, and designated it an accessory element [Ohyama, Y., Ozono, K., Uchida, M., Yoshimura, M., Shinki, T., Suda, T. and Yamamoto, O. Functional assessment of two vitamin D-responsive elements in the rat 25-hydroxyvitamin D₃ 24-hydroxylase gene. J. Biol. Chem., 1996, 271, 30381-30385]. The accessory element located adjacent to the proximal VDRE is not capable of binding to the vitamin D receptor (VDR), while its nucleotide sequence resembles the consensus sequence of VDREs, direct repeat 3 (DR3). To clarify the difference between the accessory element and VDREs, the function of the accessory element was compared with that of VDREs. The mutated accessory elements with a single nucleotide substitution showed the capability of binding to the VDR in vitro. However, these mutants still did not act as a VDRE when driven by the heterologous SV40 promoter. The accessory element did not enhance the function of a cAMP-responsive element. The corresponding site of the accessory element in the human 24-hydroxylase is a DR4-type element, and this element did not function as an accessory element. These results indicate that a critical nucleotide sequence is necessary for the binding to the VDR and for mediating the vitamin D effect, and suggest the different regulation between the rat and human 24-hydroxylase gene.     

Localization of blood-group-related linear poly-N-acetyllactosamine structure in different human tissues by Griffonia simplicifolia agglutinin-II staining following endo-β-galactosidase digestion

Summary Endo--galactosidase from Escherichia freundii cleaves polylactosaminyl structures as follows: R-GlcNAc1-3Gal1-4GlcNAc1-R + H₂O R-GlcNAc1–3Gal + GlcNAc1-R. By staining with Griffonia simplicifolia agglutinin-II following the enzyme digestion, the distribution of R-GlcNAc1–3Gal1–4GlcNAc can be demonstrated in tissue sections. This carbohydrate chain is one of the backbone structures carrying the blood-group-related antigens and, thus, localization of this structure may provide detailed information about the distribution of variants with different backbone structures. Various formalin-fixed, paraffin-embedded tissue sections were stained by Griffonia simplicifolia agglutinin-II with or without prior enzyme digestion and the reactivity of the agglutinin imparted by enzyme digestion was studied in the following tissues and cells: pancreatic acinar cells, gastric surface mucosae, duct cells and mucous cells of salivary glands and tracheal glands, surface epithelium of trachea, goblet cells of large intestine, columnar epithelium of uterine cervical glands, distal and collecting tubules of kidney, certain cells of anterior lobe and colloid of middle lobe of pituitary glands, epithelial reticular cells and Hassall's corpuscles of thymus and Kupffer cells of liver. In gastric surface mucosae, the reactivity of the agglutinin appeared in non-secretor individuals but not in the secretor individuals, and in mucous cells of salivary and tracheal glands the reactivity appeared in Le(a - b -) non-secretor individuals but not in Le(a + b -) non-secretor or secretor individuals. In pancreatic acinar cells and duct cells of salivary glands from fetuses and newborn infants, prior fucosidase digestion markedly enhanced the Griffonia simplicifolia agglutinin-II reactivity elicited by endo--galactosidase digestion. Prior fucosidase digestion was also a prerequisite for revealing the reactivity of this agglutinin by endo--galactosidase digestion in gastric surface mucosae from secretor individuals. -Galactosidase digestion disclosed reactivity of this agglutinin in pancreatic acinar cells and duct cells of salivary glands even after the removal of endo--galactosidase-labile lactosamine structures by sequential digestion with endo--galactosidase and -N-acetylhexosaminidase. These results demonstrate that the procedures developed in this study provide a useful means for detecting different types of lactosamine structures which carry blood-group antigens in humans tissues.     

A facile transphosphatidylation reaction using a culture supernatant of actinomycetes directly as a phospholipase D catalyst with a chelating agent

An attempt was made to use the phospholipase D (PLD)- containing culture supernatants of actinomycetes directly as catalysts for the transphosphatidylation reaction of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in a biphasic system. Of the five actinomycetes (three Streptomyces sp. and two Streptoverticillium sp.) examined, three (St. mediocidicus, Stv. cinnamoneum and Stv. hachijoense) exhibited good PLD production performance, but the selectivity (ratio of transphosphatidylation to hydrolysis) of the PLDs in the culture supernatant of all three actinomycetes were significantly low. However, the addition of EDTA to the reaction mixture as a chelating agent remarkably improved the selectivity of the PLDs, which approached 100% in all the culture supernatants. Commercially available PLDs were also investigated and classified into two types. The PLDs of one type had high selectivity and no metal was required for the enzyme activity, while those of the other type showed low selectivity and a metal was necessary for the enzyme to be activated. From this finding, it was considered that the culture supernatants used in this study contained several PLDs of both types. When the chelating agent was added to the reaction mixture, the hydrolysis due to PLDs with low selectivity was suppressed by removal of the essential metal, resulting in an increased in the overall selectivity of the PLDs in the culture supernatant. Repeated batch transphosphatidylation reactions were performed 20 times, reusing the PLDs in the aqueous phase by centrifugation; the reaction rate gradually decreased to 60% of that of batch 1 by batch 20. This suggests that the transphosphatidylation reaction using a culture supernatant has potential for industrial application. (c) 1994 John Wiley & Sons, Inc.     

Stereochemistry during aflatoxin biosynthesis: conversion of norsolorinic acid to averufin.

A reaction sequence, norsolorinic acid (NA)-->averantin (AVN)-->5'-hydroxyaverantin (HAVN)-->averufin (AVR), is the early part of a biosynthetic pathway for aflatoxins. In this study, we determined the stereochemical relationship among these metabolites by using chiral high-performance liquid chromatography. In cell-free experiments using the cytosol fraction of Aspergillus parasiticus NIAH-26, (1'S)-AVN was exclusively produced from NA in the presence of NADPH. Also, only (1'S)-AVN, and not (1'R)-AVN, served as a substrate for the reverse reaction from AVN to NA. When the microsome fraction of NIAH-26 was incubated with (1'S)-AVN in the presence of NADPH, two HAVN diastereomers and one AVR enantiomer were formed, whereas these substances were never produced from (1'R)-AVN. Moreover, (1'S,5'R)-AVR was exclusively formed from both HAVN diastereomers by the cytosol fraction in the presence of NAD. The feeding experiments using this mutant showed that aflatoxins were produced from (1'S,5'R)-AVR but not from (1'R,5'S)-AVR. These results indicate that the enzymes involved in this pathway show strict stereospecificity to their substrates and that the configuration of (1'S,5'R)-AVR leading to the formation of aflatoxins is due to the stereospecificity of NA dehydrogenase which catalyzes the reaction between (1'S)-AVN and NA.