Pharmacy responses during the COVID-19 pandemic: a questionnaire survey.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has heavily affected the economy, industries, and medicine. Local governments and medical institutions have struggled to respond. The purpose of this questionnaire survey was to evaluate strategies for pharmacy services, availability of ethanol for disinfection, and measures adopted for in-house infection control aiming to enhance future infection control efforts. METHODS: Since pharmacies have been also affected by the COVID-19 pandemic, we surveyed COVID-19 measures taken at 174 pharmacies in Ehime prefecture, Japan. RESULTS: The survey showed that pharmacies made changes to facilities and equipment, such as installing partitions at dispensing counters, procuring personal protective equipment for employees, and using ethanol for disinfection, even when these items were in short supply. Pharmacies also adopted new strategies, such as holding meetings with suppliers and internal staff via online platforms. Many pharmacies also undertook COVID-19 preventive measures, such as preparing documentation of infection control measures and disinfectants. Moreover, they held lectures and workshops on disinfection and infection control measures. CONCLUSIONS: From public health perspectives, pharmacies should adopt measures to prevent infections spread and, if necessary, utilise online tools and other new strategies to achieve this goal. It is also essential to educate the public about infection control, stockpile supplies, and work with hospitals to prevent COVID-19 spreads.

LacdiNAcylation of N-glycans in MDA-MB-231 human breast cancer cells results in changes in morphological appearance and adhesive properties of the cells.

We demonstrated previously that the expression of the disaccharide, GalNAcß1 → 4GlcNAc (LacdiNAc), on N-glycans of cell surface glycoproteins in MDA-MB-231 human breast cancer cells suppresses their malignant properties such as tumor formation in nude mice. Here, we report changes in the morphological appearance and adhesive properties of two kinds of clonal cells of MDA-MB-231 cells overexpressing ß4-N-acetyl-galactosaminyltransferase 4. The clonal cells exhibited a cobble stone-like shape as compared to a spindle-like shape of the mock-transfected cells and the original MDA-MB-231 cells. This was associated with an increased expression of cell surface E-cadherin, a marker of epithelial cells, and a decreased expression of N-cadherin, vimentin, α-smooth muscle actin and ZEB1, markers of mesenchymal cells. In addition, the clonal cells showed a lower migratory activity compared to the mock-transfected cells by wound-healing assay. These results suggest that mesenchymal-epithelial transition may be occurring in these clonal cells. Furthermore, increased adhesion to extracellular matrix proteins such as fibronectin, collagen type I, collagen type IV, and laminin was observed. The clonal cells spread and enlarged, whereas the mock-transfected cells demonstrated poor spreading on laminin-coated plates in the absence of fetal calf serum, indicating that expression of LacdiNAc on cell surface glycoproteins results in changes in cell adhesive and spreading properties particularly to laminin.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

BACKGROUND: Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. RESULTS: By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. CONCLUSIONS: We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Isolation of a methylated mannose-binding protein from terrestrial worm Enchytraeus japonensis.

To elucidate a biological role of the methylated mannose residues found in N-glycans of terrestrial worm Enchytraeus japonensis, we first synthesized 3-O-methyl mannose- and 4-O-methyl mannose-derivatives and immobilized them to Sepharose 4B beads in order to isolate the sugar-binding protein. When whole protein extracts from the worms was applied to a series of the columns immobilized with the modified and unmodified mannose-derivatives, respectively, a protein with a molecular weight of 25,000 was isolated by 4-O-methyl mannose-immobilized column chromatography, and termed as a methylated mannose-binding protein (mMBP). mMBP bound weakly to a mannose-immobilized column and moderately to a 3-O-methyl mannose-immobilized column. The N-terminal amino acid sequences of mMBP and its endoprotease-digested peptides were determined. Using the degenerate first primers synthesized based on the primary sequence, a genomic DNA fragment was isolated. Then, the second primers were synthesized based on the genomic DNA fragment, and with use of them two cDNA fragments were obtained by the 3'- and 5'-RACE methods. Finally, the third primers were synthesized based on the sequences of the two cDNA fragments and one genomic DNA fragment, and with use of them a full-length cDNA of mMBP was isolated and shown to comprise a putative 633 bp open reading frame encoding 210 amino acid residues. BLAST analysis revealed that mMBP has identities by 26 ~ 55% to several proteins including the regeneration-upregulated protein 3 from the same species. Whether mMBP is involved in the regeneration of the worm is under investigation.

Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans.

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.

Preferential binding of E. coli with type 1 fimbria to D-mannobiose with the Manα1 → 2Man structure.

Manα1 → 2Man, Manα1 → 3Man, Manα1 → 4Man, and Manα1 → 6Man were converted to the glycosylamine derivatives. Then, they were mixed with monobenzyl succinic acid to obtain their amide derivatives. After removing the benzyl group by hydrogenation, the succinylamide derivatives were coupled with the hydrazino groups on BlotGlyco™ beads in the presence of water-soluble carbodiimide. d-Mannobiose-linked beads were incubated with fluorescence-labeled Escherichia coli with type 1 fimbria, and the number of the fluorescent dots associated with the beads was counted in order to determine the binding preference among d-mannobiose isomers. The results showed that the bacteria bind strongly to Manα1 → 2Man1 → beads, Manα1 → 3Man1 → beads, Manα1 → 4Man1 → beads, and Manα1 → 6Man1 → beads, in order. In the presence of 0.1 M methyl α-d-mannopyranoside, most of the bacteria failed to bind to these beads. These results indicate that E. coli with type 1 fimbria binds to all types of d-mannobiose isomers but preferentially to Manα1 → 2Man disaccharide.

Enhanced expression of the ß4-N-acetylgalactosaminyltransferase 4 gene impairs tumor growth of human breast cancer cells.

Two ß4-N-acetylgalactosaminyltransferases (ß4GalNAcTs), ß4GalNAcT3 and ß4GalNAcT4, have been shown to be involved in the synthesis of the GalNAcß1 â†’ 4GlcNAc (LacdiNAc) group expressed on the outer branches of N- and/or O-glycans, and only ß4GalNAcT4 is expressed in human mammary gland. We found that the expression level of the LacdiNAc group decreases as human breast cancers progress. To investigate biological significances of this disaccharide in human breast cancers, we transfected the FLAG-tagged ß4GalNAcT4 cDNA into MDA-MB-231 cells, and obtained several clones showing enhanced expression of the gene. Clones 1 and 2 showed 15 and 9 times more transcript than mock-transfected cells. The FLAG-ß4GalNAcT4 protein and its product, the LacdiNAc group, were detected in clone 1 and 2 cells. No change was observed in their growth rates while significant decreases in colony forming and invasive abilities were observed for clone 1 and 2 cells. When clone 1 cells were transplanted subcutaneously into nude mice, no tumors were formed while tumors were formed with mock-transfected cells. These results indicate that the expression of the LacdiNAc group is quite important for the suppression of malignancies of the MDA-MB-231 cells.

Challenge to the suppression of tumor growth by the ß4-galactosyltransferase genes.

It has been well established that structural changes in glycans attached to proteins and lipids are associated with malignant transformation of cells. We focused on galactose residues among the sugars since they are involved in the galectin-mediated biology, and many carbohydrate antigens are frequently expressed on this sugar. We found changes in the expression of the ß4-galactosyltransferase (ß4GalT) 2 and 5 genes in cancer cells: decreased expression of the ß4GalT2 gene and increased expression of the ß4GalT5 gene. The growth of mouse melanoma cells showing enhanced expression of the ß4GalT2 gene or reduced expression of the ß4GalT5 gene is inhibited remarkably in syngeneic mice. Tumor growth inhibition is probably caused by the induction of apoptosis, inhibition of angiogenesis, and/or reduced MAPK signals. Direct transduction of human ß4GalT2 cDNA together with the adenovirus vector into human hepatocellular carcinoma cells grown in SCID mice results in marked growth retardation of the tumors. ß4GalT gene-transfer appears to be a potential tool for cancer therapy.

Gene expression levels of ß4-galactosyltransferase 5 correlate with the tumorigenic potentials of B16-F10 mouse melanoma cells.

Our previous studies showed that mouse ß4-galactosyltransferase 5 (ß4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the ß4GalT5 gene. We isolated a stable clone named E5 whose ß4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous ß4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the ß4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by ß4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

Expression and characterization of the first snail-derived UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41) catalyzes the first step in mucin-type O-glycosylation. To date, several members of this large enzyme family have been analyzed in detail. In this study we present cloning, expression and characterization of the first representative of this type of glycosyltransferase from mollusk origin, namely from Biomphalaria glabrata. The full length sequence of the respective gene was obtained by screening of a cDNA library using homology-based PCR. The entire gene codes for a protein consisting of 600 amino acids comprising the features of a typical type II membrane protein containing a cytoplasmic tail at the N-terminus, a transmembrane and a catalytic domain as well as a ricin-like motif at the C-terminus. Sequence comparison with ppGalNAcTs from various species revealed high similarities in terms of structural architecture. The enzyme is O-glycosylated but does not have any putative N-glycosylation sites. All four tested acceptor peptides were functional substrates, with Muc2 being the best one. Further biochemical parameters tested, confirmed a close relationship to the family of yet known ppGalNAcTs.

Biosynthesis and Biological Significances of LacdiNAc Group on N- and O-Glycans in Human Cancer Cells.

An increasing number of studies have shown that the disaccharide GalNAcß1→4GlcNAc (LacdiNAc) group bound to N- and O-glycans in glycoproteins is expressed in a variety of mammalian cells. Biosynthesis of the LacdiNAc group was well studied, and two ß4-N-acetylgalactosaminyltransferases, ß4GalNAcT3 and ß4GalNAcT4, have been shown to transfer N-acetylgalactosamine (GalNAc) to N-acetylglucosamine (GlcNAc) of N- and O-glycans in a ß-1,4-linkage. The LacdiNAc group is often sialylated, sulfated, and/or fucosylated, and the LacdiNAc group, with or without these modifications, is recognized by receptors and lectins and is thus involved in the regulation of several biological phenomena, such as cell differentiation. The occurrences of the LacdiNAc group and the ß4GalNAcTs appear to be tissue specific and are closely associated with the tumor progression or regression, indicating that they will be potent diagnostic markers of particular cancers, such as prostate cancer. It has been demonstrated that the expression of the LacdiNAc group on N-glycans of cell surface glycoproteins including ß1-integrin is involved in the modulation of their protein functions, thus affecting cellular invasion and other malignant properties of cancer cells. The biological roles of the LacdiNAc group in cancer cells have not been fully understood. However, the re-expression of the LacdiNAc group on N-glycans, which is lost in breast cancer cells by transfection of the ß4GalNAcT4 gene, brings about the partial restoration of normal properties and subsequent suppression of malignant phenotypes of the cells. Therefore, elucidation of the biological roles of the LacdiNAc group in glycoproteins will lead to the suppression of breast cancers.

Comprehensive analysis of mitochondria in roots and hypocotyls of soybean under flooding stress using proteomics and metabolomics techniques.

Flooding is a serious problem for soybeans because it reduces growth and grain yield. Proteomic and metabolomic techniques were used to examine whether mitochondrial function is altered in soybeans by flooding stress. Mitochondrial fractions were purified from the roots and hypocotyls of 4-day-old soybean seedlings that had been flooded for 2 days. Mitochondrial matrix and membrane proteins were separated by two-dimensional polyacrylamide gel electrophoresis and blue-native polyacrylamide gel electrophoresis, respectively. Differentially expressed proteins and metabolites were identified using mass spectrometry. Proteins and metabolites related to the tricarboxylic acid cycle and γ-amino butyrate shunt were up-regulated by flooding stress, while inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were down-regulated. The amounts of NADH and NAD were increased; however, ATP was significantly decreased by flooding stress. These results suggest that flooding directly impairs electron transport chains, although NADH production increases in the mitochondria through the tricarboxylic acid cycle.

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression.

Deficiency of Smad3, an intracellular mediator of TGF-ß, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3(ex8/ex8) C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3(-/-) mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3(-/-) mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear ß-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3(-/-) mice in accordance with nuclear ß-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3(-/-) mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3(-/-) mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a "proliferative zone" at the bottom of colonic crypts in the normal colon.

Involvement of Galectin-3 with vascular cell adhesion molecule-1 in growth regulation of mouse BALB/3T3 cells.

beta-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the beta-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal beta-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean beta-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.

Involvement of murine ß-1,4-galactosyltransferase V in lactosylceramide biosynthesis.

Human ß-1,4-galactosyltransferase (ß-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from ß-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the ß-1,4-GalT I-VI genes revealed that the expression of the ß-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other ß-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When ß-1,4-GalT activities were determined towards GlcNAcß-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine ß-1,4-GalT V is involved in Lac-Cer biosynthesis.

Comparative proteomics analysis of differentially expressed proteins in soybean cell wall during flooding stress.

Flooding is a major problem for soybean crop as it reduces the growth and grain yield. To investigate the function of the soybean cell wall in the response to flooding stress, cell wall proteins were analyzed. Cell wall proteins from roots and hypocotyls of soybeans, which were germinated for 2 days and subjected to 2 days of flooding, were purified, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Sixteen out of 204 cell wall proteins showed responses to flooding stress. Of these, two lipoxygenases, four germin-like protein precursors, three stem 28/31 kDa glycoprotein precursors, and one superoxide dismutase [Cu-Zn] were downregulated. A copper amine oxidase was found to have shifted from the basic to acidic zone following flooding stress. Based on these results, it was confirmed by the lignin staining that the lignification was suppressed in the root of soybean under the flooding stress. These results suggest that the roots and hypocotyls of soybean caused the suppression of lignification through decrease of these proteins by downregulation of reactive oxygen species and jasmonate biosynthesis under flooding stress.

Identification of flooding stress responsible cascades in root and hypocotyl of soybean using proteome analysis.

Flooding inducible proteins were analyzed using a proteomic technique to understand the mechanism of soybean response to immersion in water. Soybeans were germinated for 2 days, and then subjected to flooding for 2 days. Proteins were extracted from root and hypocotyl, separated by two-dimensional polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue, and analyzed by protein sequencing and mass spectrometry. Out of 803 proteins, 21 proteins were significantly up-regulated, and seven proteins were down-regulated by flooding stress. Of the total, 11 up-regulated proteins were classified as related to protein destination/storage and three proteins to energy, while four down-regulated proteins were related to protein destination/storage and three proteins to disease/defense. The expression of 22 proteins significantly changed within 1 day after flooding stress. The effects of flooding, nitrogen substitution without flooding, or flooding with aeration were analyzed for 1-4 days. The expression of alcohol dehydrogenase increased remarkably by nitrogen substitution compared to flooding. The expression of many proteins that changed due to flooding showed the same tendencies observed for nitrogen substitution; however, the expression of proteins classified into protein destination/storage did not.

Analysis of plasma membrane proteome in soybean and application to flooding stress response.

The plasma membrane acts as the primary interface between the cellular cytoplasm and the extracellular environment. To investigate the function of the plasma membrane in response to flooding stress, plasma membrane was purified from root and hypocotyl of soybean seedlings using an aqueous two-phase partitioning method. Purified plasma membrane proteins with 81% purity were analyzed using either two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry and protein sequencing (2-DE MS/sequencer)-based proteomics or nanoliquid chromatography followed by mass spectrometry (nanoLC-MS/MS)-based proteomics. The number of hydrophobic proteins identified by nanoLC-MS/MS-based proteomics was compared with those identified by 2-DE MS/sequencer-based proteomics. These techniques were applied to identify the proteins in soybean that are responsive to flooding stress. Results indicate insights of plasma membrane into the response of soybean to flooding stress: (i) the proteins located in the cell wall are up-regulated in plasma membrane; (ii) the proteins related to antioxidative system play a crucial role in protecting cells from oxidative damage; (iii) the heat shock cognate protein plays a role in protecting proteins from denaturation and degradation during flooding stress; and (iv) the signaling related proteins might regulate ion homeostasis.

Early lethality of beta-1,4-galactosyltransferase V-mutant mice by growth retardation.

The beta-1,4-galactosyltransferase (beta-1,4-GalT) V whose human and mouse genes were cloned by us has been suggested to be involved in the biosynthesis of N-glycans and O-glycans, and lactosylceramide. To determine its biological function, beta-1,4-GalT V (B4galt5) mutant mice obtained by a gene trap method were analyzed. Analysis of pre- and post-implantation embryos revealed that the B4galt5(-/-) mice die by E10.5 while B4galt5(+/-) mice were born and grown normally. Histological study showed that most tissues are formed in B4galt5(-/-) embryos but their appearance at E10.5 is close to that of B4galt5(+/-) embryos at E9.0-9.5. The results indicate that the growth is delayed by one to one and half day in B4galt5(-/-) embryos when compared to B4galt5(+/-) embryos, which results in early death of the embryos by E10.5, probably due to hematopoietic and/or placental defects.