Learning/Memory Impairment and Reduced Expression of the HNK-1 Carbohydrate in ��4-Galactosyltransferase-II-deficient Mice

The glycosylation of glycoproteins and glycolipids is important for central nervous system development and function. Although the roles of several carbohydrate epitopes in the central nervous system, including polysialic acid, the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides, have been investigated, those of the glycan backbone structures, such as Galβ1-4GlcNAc and Galβ1-3GlcNAc, are not fully examined. Here we report the generation of mice deficient in β4-galactosyltransferase-II (β4GalT-II). This galactosyltransferase transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the Gal β1-4GlcNAc structure, and it is strongly expressed in the central nervous system. In behavioral tests, the β4GalT-II^-/- mice showed normal spontaneous activity in a novel environment, but impaired spatial learning/memory and motor coordination/learning. Immunohistochemistry showed that the amount of HNK-1 carbohydrate was markedly decreased in the brain of β4GalT-II^-/- mice, whereas the expression of polysialic acid was not affected. Furthermore, mice deficient in glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of the HNK-1 carbohydrate, also showed impaired spatial learning/memory as described in our previous report, although their motor coordination/learning was normal as shown in this study. Histological examination showed abnormal alignment and reduced number of Purkinje cells in the cerebellum of β4GalT-II^-/- mice. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that the glycans synthesized by β4GalT-II have essential roles in higher brain functions, including some that are HNK-1-dependent and some that are not.The glycosylation of glycoproteins, proteoglycans, and glycolipids is important for their biological activities, stability, transport, and clearance from circulation, and cell-surface glycans participate in cell-cell and cell-extracellular matrix interactions. In the central nervous system, several specific carbohydrate epitopes, including polysialic acid (PSA),³ the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and oligomannosides play indispensable roles in neuronal generation, cell migration, axonal outgrowth, and synaptic plasticity (1). Functional analyses of the glycan backbone structures, like lactosamine core (Galβ1-4GlcNAc), neolactosamine core (Galβ1-3GlcNAc), and polylactosamine (Galβ1-4GlcNAcβ1-3) have been carried out using gene-deficient mice in β4-galactosyltransferase-I (β4GalT-I) (2, 3), β4GalT-V (4), β3-N-acetylglucosaminyl-transferase-II (β3GnT-II) (5), β3GnT-III (Core1-β3GnT) (6), β3GnT-V (7), and Core2GnT (8). However, the roles of these glycan backbone structures in the nervous system have not been examined except the olfactory sensory system (9).β4GalTs synthesize the Galβ1-4GlcNAc structure via the β4-galactosylation of glycoproteins and glycolipids; the β4GalTs transfer galactose (Gal) from UDP-Gal to a nonreducing terminal N-acetylglucosamine (GlcNAc) of N- and O-glycans with a β-1,4-linkage. The β4GalT family has seven members (β4GalT-I to VII), of which at least five have similar Galβ1-4GlcNAc-synthesizing activities (10, 11). Each β4GalT has a tissue-specific expression pattern and substrate specificity with overlapping, suggesting each β4GalT has its own biological role as well as redundant functions. β4GalT-I and β4GalT-II share the highest identity (52% at the amino acid level) among the β4GalTs (12), suggesting these two galactosyltransferases can compensate for each other. β4GalT-I is strongly and ubiquitously expressed in various non-neural tissues, whereas β4GalT-II is strongly expressed in neural tissues (13, 14). Indeed, the β4GalT activity in the brain of β4GalT-I-deficient (β4GalT-I^-/-) mice remains as high as 65% of that of wild-type mice, and the expression levels of PSA and the HNK-1 carbohydrate in the brain of these mice are normal (15). These results suggest β4GalTs other than β4GalT-I, like β4GalT-II, are important in the nervous system.Among the β4GalT family members, only β4GalT-I^-/- mice have been examined extensively; this was done by us and another group. We reported that glycans synthesized by β4GalT-I play various roles in epithelial cell growth and differentiation, inflammatory responses, skin wound healing, and IgA nephropathy development (2, 16-18). Another group reported that glycans synthesized by β4GalT-I are involved in anterior pituitary hormone function and in fertilization (3, 19). However, no other nervous system deficits have been reported in these mice, and the role of the β4-galactosylation of glycoproteins and glycolipids in the nervous system has not been fully examined.In this study, we generated β4GalT-II^-/- mice and examined them for behavioral abnormalities and biochemical and histological changes in the central nervous system. β4GalT-II^-/- mice were impaired in spatial learning/memory and motor coordination/learning. The amount of HNK-1 carbohydrate was markedly decreased in the β4GalT-II^-/- brain, but PSA expression was not affected. These results suggest that the Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-II and that glycans synthesized by β4GalT-II have essential roles in higher brain functions, including ones that are HNK-1 carbohydrate-dependent and ones that are independent of HNK-1.     

Phthalocyanine-dyed fibers adsorb allergenic proteins

Summary. Phthalocyanine (Pc)-dyed fiber is reported to reduce atopic symptoms in some patients when they use underwear made of the fiber. We investigated the adsorption of allergens on Pc-fiber. Pc-fiber trapped house dust/pollen/food allergens with varied molecular weight and pI. The adsorbed allergens were released in the presence of mild detergent. Pc-fiber did not change the molecular weight or disulfide bonding of the allergens. These observations imply that Pc-fiber is applicable as an “allergen trap” for a wide variety of products.     

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen

A group of rat monoclonal antibodies recognizing the six different chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.     

Inhibition of pancreatic and microbial lipases by proteins

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Computer analysis of sub-parameters of depurination and depolymerization rate constants in Feulgen DNA hydrolysis

Feulgen DNA hydrolysis curves derived from cytofluorometry at various temperatures and HCl concentrations were computer analyzed with least squares fit to Bateman function. By comparing the depurination (k1) and depolymerization (k2) rate constants at different hydrolysis conditions, it was found that the two parameters of temperature and HCl concentration can be expressed as k = AN2 X exp (-B/T), where A and B are constants, N is the HCl concentrations, and T is the absolute temperature. From the analysis of Feulgen hydrolysis curves with 2N HCl at various temperatures, it was calculated that A = 5.3590 X 10(14) and B = 12133.543, for k1, and A = 6.2401 X 10(14) and B = 12181.660, for k2 for mouse 4C hepatocytes fixed with absolute methanol. Computer generated theoretical hydrolysis curves using the above k1 and k2 values were compared with experimental curves at various temperatures and HCl-concentrations. The two types of hydrolysis curves coincided with each other when 1-3 N HCl was used at temperatures between 30-40 degrees C. The peak times of hydrolysis curves at different conditions determined by experimental analysis and theoretical estimations also coincided reasonably well with each other. The physico-chemical phenomena underlying the equation designating k1 and k2 values are discussed.     

Comparative study of Agkistrodon acutus venoms from China and Taiwan

Three hemorrhagic toxins (Ac1-, Ac2- and Ac3-proteinases) were isolated from the lyophilized venom of Agkistrodon acutus from China using gel filtration on a Sephadex G-75 column, followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A-50, DEAE-Sephacel and DEAE-cellulose. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis. Three hemorrhagic toxins possessed both lethal and proteolytic activities. These activities were inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol (beta-aminoethyl)N,N,N',N'-tetraacetic acid (EGTA), o-phenanthroline or cysteine, but not by soybean trypsin inhibitor (SBTI), p-chloromercuribenzoate (PCMB) or diisopropyl fluorophosphate (DFP). Molecular weight of hemorrhagic toxins (Ac1-, Ac2- and Ac3-proteinases) were determined to be 24,500, 25,000 and 57,000, respectively. It was found that hemorrhagic toxins have considerable similarity to hemorrhagic toxins isolated by Nikai et al. (1977) and Sugihara et al. (1978, 1979), but Ac2- and Ac3-proteinases from China and Taiwan are electrophoretically unrelated.