Biochemical studies on a case of feline mannosidosis.

Evidence is presented for the biochemical diagnosis of the first case of feline mannosidosis. A marked deficiency of acidic alpha-D-mannosidase in the brain, kidney and liver and excessive excretion of mannose-rich oligosaccharides in the urine were found in a kitten suffering from a nervous disorder. Residual acidic alpha-D-mannosidase, ranging from 2 to 5.5% of the normal activity, was observed in the tissues of the affected kitten. It has similar kinetic and physicochemical properties to the normal activity. The amount of mannose in the urine of the affected kitten was 19-fold greater than in a comparable control, and the molar ratio of mannose to N-acetylglucosamine was approx. 6 : 1. High concentrations of neutral oligosaccharides were detected in the urine. The predominant oligosaccharide appeared to be a hexasaccharide. The biochemical features of bovine, feline and human mannosidosis are compared, and it is concluded that feline mannosidosis may be a useful animal model for studying the human disease.     

Chromatographic separation of oligosaccharides from mannosidosis urine

A mixture of oligosaccharides was isolated from mannosidosis urine by a rapid and convenient procedure employing adsorption on activated charcoal. The mixture was partially fractionated into a homologous series of compounds by a rapid procedure employing a preparative, liquid chromatograph, and a more complete separation was obtained by a second chromatographic step employing a solid phase having more-powerful resolving properties, or by preparative-layer chromatography. The series of oligosaccharides was completely separated by 7-MPa, liquid chromatography (l.c.) on a Micropak NH₂-10 column; the analysis could be performed with isocratic or gradient solvent-systems, and did not involve derivatization. With the isocratic system, a strict relationship was observed between retention time and the number of d-mannosyl residues. The use of 1,4-diaminobutane as a column “restorer” was evaluated.     

Structural studies of urinary oliogosaccharides from patients with mannosidosis

Eight neutral oligosaccharide fractions were obtained from the pooled urine of two patients with mannosidosis by Bio-Gel P2 and Bio-Gel P4 column chromatography. The structures of seventeen oligosaccharides were determined by monosaccharide composition analysis, methylation studies, acetolysis, Smith degradation, and ¹³C NMR analysis. Three of the proposed structures, Manα1-3Manβ1-4GlcNAc, Manα1-2Manα1-3Manβ1-4GlcNAc, and Manα1-2Manα1-2Manα1-3Manβ1-4GlcNAc are identical to those first published by Norden et al. (N. E. Norden, A. Lundblad, S. Svennson, P. A. Ockerman, and S. Autio, 1973. J. Biol. Chem.248, 6210–6215; N. E. Norden, A. Lundblad, S. Svennson, and S. Autio, 1974. Biochemistry13, 871–874). Thirteen of them, Manα1-3Manα1-6(Manα1-3)-Manβ1-4GlcNAc, Manα1-3Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAc, and 11 isomers of (Manα1-2)_0–4[Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc], are the same as those first published by Yamashita et al. (K. Yamashita, Y. Tachibana, K. Mihara, S. Okada, H. Yabuuchi, and A. Kobata, 1980, J. Biol. Chem.255, 5126–5133); a tetrasac-charide, Manα1-6(Manα1-3)Manβ1-4GlcNAc, is newly reported and several other structural possibilities are proposed.     

The catabolism of mammalian glycoproteins. Comparison of the storage products in bovine, feline and human mannosidosis.

Analysis of the neutral urinary oligosaccharides in bovine, feline and human mannosidosis by thin-layer and gel-permeation chromatography has shown that the patterns of stored oligosaccharides in the three species are different. In bovine and feline mannosidosis the most abundant urinary oligosaccharide is also the most abundant in the tissues of each species. The predominant oligosaccharides were purified by a combination of gel-filtration, ion-exchange and thin-layer chromatography and shown to contain only mannose and N-acetylglucosamine by g.l.c. and g.l.c.--mass spectrometry. The probable composition and size of each oligosaccharide were predicted from its chromatographic properties, sugar composition and the known structure of asparagine-linked oligosaccharides. The bovine and feline oligosaccharides belonged to a homologous series of general composition Mann (GlcNAc)2, whereas the human oligosaccharides belong to a different series, MannGlcNAc. These structures suggest that lysosomal endohexosaminidase is not present in bovine and feline tissues. The predominant feline storage product, Man3(GlcNAc)2, was the expected storage product from the catabolism of complex asparagine-linked glycans. In contrast, the predominant bovine oligosaccharide, Man2(GlcNAc)2, probably lacks one of the alpha-linked mannose residues in the core region. A similar situation occurs in human mannosidosis. It is predicted that in these species either that the residual mutant alpha-D-mannosidase retains activity towards one of the core alpha-linked mannose residues or that another form of lysosomal alpha-D-mannosidase that is unaffected in these disorders occurs. It is concluded that the differences in storage products are due to differences in the catabolic pathways of glycoproteins among the species.     

System beta and system A amino acid transporters in the feline endotheliochorial placenta

There is no knowledge of the transport mechanisms by which solutes cross the cat placenta or any other endotheliochorial placenta. Here, we investigated whether the amino acid transport systems beta and A are present in the cat placenta using a placental fragment uptake technique. Data were compared with studies in the human placenta, in which the presence of these two transport systems has been well established. A time course of [(3)H]taurine (substrate for system beta) and [(14)C]MeAIB (nonmetabolizable substrate for system A) uptake was determined in the term cat and human placental fragments in the presence and absence (choline substituted) of Na(+), and further studies were carried out over 15 min. Taurine uptake into both cat and human placenta fragments was found to be Na(+) and Cl(-) dependent, and Na(+)-dependent taurine uptake was blocked by excess beta-alanine. MeAIB uptake was found to be Na(+) dependent, and Na(+)-dependent MeAIB uptake was blocked by excess MeAIB or glycine. Western blotting and immunohistochemistry performed on cat and human placenta showed expression of TAUT and ATA2 (SNAT2), proteins associated with system beta and system A activity, respectively. This study therefore provides the first evidence of the presence of amino acid transport systems beta and A in the cat placenta.     

Specificity studies on alpha-mannosidases using oligosaccharides from mannosidosis urine as substrates.

Oligosaccharides containing terminal non-reducing alpha(1 leads to 2)-, alpha(1 leads to 3)-, and alpha(1 leads to 6)-linked mannose residues, isolated from human and bovine mannosidosis urines were used as substrates to test the specificities of acidic alpha-mannosidases isolated from human and bovine liver. The enzymes released all the alpha-linked mannose residues from each oligosaccharide and were most effective on the smallest substrate. Enzyme A in each case was less active on the oligosaccharides than alpha-mannosidase B2, even though the apparent Km value for the substrates was the same with each enzyme. The human acidic alpha-mannosidases were also found to be more active on substrates isolated from human rather than bovine mannosidosis urine. Human alpha-mannosidase C, which has a neutral pH optimum when assayed with a synthetic substrate, did not hydrolyse any of the oligosaccharides at neutral pH, but was found to be active at an acidic pH.     

Antemortem diagnosis of an apparent case of feline candidiasis

Candidiasis in cats has always been linked with such predisposing factors as parvovirus infections and antibiotic and chemotherapeutic treatments. Moreover these cases were all diagnosed post-mortem.The clinical observations and the diagnostic procedures used in an antemortem case of probable idiopathic intestinal candidiasis in a cat are reported. The therapeutic measures used and the method of evaluating the efficacy of antimycotic treatment are also described.     

Oligosaccharides on cathepsin D from porcine spleen

Cathepsin D from porcine spleen contained mannose (3.3%), glucosamine (1.4%), and mannose 6-phosphate (0.08%). Essentially all of the oligosaccharides of cathepsin D could be released by endo-β-N-acetylglucosaminidase H, pointing to oligomajmoside types of structures. Three neutral oligosaccharide fractions, containing 5, 6, and 7 mannose residues, respectively, were isolated by gel permeation chromatography on Bio-Gel P-2. Studies using exoglycosidase digestions and 500-MHz ¹H NMR spectroscopy revealed that their structures are [Manα1 → 2]_{0 or 1}Manα1 → 6[Manα1 → 3]Manα1 → 6[(Manα1 → 2)_{0 or 1}Manα1 → 3]Manβ1 → 4GlcNAcβ1 → 4 GlcNAc. These structures are identical to what have recently been proposed by Takahashi et al. for the major oligosaccharide units of cathepsin D from the same source (T. Takahashi P.G. Schimidt, and J. Tang (1983)J. Biol. Chem.258, 2819–2930), except for the occurrence of two isomeric oligosaccharides containing six mannoses. Only a part (3.4%) of the oligosaccharides were acidic, containing phosphates in monoester linkage. The phosphorylated oligosaccharides also consisted of oligomannoside-type chains which were analogous to, but more heterogeneous in size than the neutral oligosaccharides. Cathepsin D was bound to a mannose- and N-acetylglucosamine-specific lectin (mannan-binding protein) isolated from rabbit liver with the K_i value of 5.4 × 10^?6m.     

Lung surfactant proteins in the early human placenta

Pulmonary surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). The biological functions of SP-A and SP-D are primarily twofold, namely surfactant homeostasis and host defense. The hydrophobic surfactant proteins, SP-B and SP-C, are required for achieving the optimal surface tension reducing properties of surfactant by promoting the rapid adsorption of surfactant phospholipids along the alveolar surface. Despite the promising findings, only little is known about the extrapulmonary distribution of these proteins. Therefore, in this study, the presence of SP-A, SP-B, SP-C and SP-D in early human placenta has been investigated. First-trimester placental tissues (22–56 days) were obtained from women undergoing curettage during normal pregnancies. In parallel tissue sections, vimentin, cytokeratin-7 and CD-68 immunostainings were used for the identification of mesenchymal cells, trophoblast cells and Hofbauer cells, respectively. According to immunohistochemistry (IHC) results, SP-A, SP-B, SP-C and SP-D immunoreactivities with different staining intensities were observed in trophoblastic layers of chorionic villous tree, trophoblastic cell columns, stromal cells, Hofbauer cells, angiogenic cell cords and vascular endothelium. Fetal hematopoietic cells showed a variable staining pattern for all four surfactant proteins ranging from none to strong intensity. Western blotting of tissue extracts confirmed our IHC results. The presence of surfactant glycoproteins in early human placenta may yield a very important feature of surfactants during first trimester and enables further studies of the role of surfactants in various pregnancy complications.     

Electron ionization mass spectra of reduced and permethylated urinary oligosaccharides from patients with mannosidosis

The electron ionization mass spectra of reduced and permethylated isomeric mixtures of the major urinary tri- to deca-oligosaccharides of patients with mannosidosis are reported. Many of the oligosaccharide isomers can be differentiated in the mixtures on the basis of their distinct fragmentation patterns.     

Isolation and characterization of a 94,000-dalton protein with thyrotropic activity from early bovine placenta

A thyrotropic protein was extracted and purified from the placenta of early bovine gestations. After protein extraction, the 45-60% ammonium sulfate precipitate of maternal and fetal bovine cotyledons was found to compete with thyroid stimulating hormone (TSH) for binding to thyroid cell membranes and to mediate TSH specific biological effects including the stimulation of cyclic AMP production, iodide uptake, and thyroxine secretion. The placental thyrotropin was further purified by gel and anion exchange chromatography, followed by binding to thyroid cell membranes and elution by mild acid treatment. 400 micrograms of isolated protein with 4.5 units of TSH-like binding activity/mg of protein was recovered from the placenta of a 90-day-old bovine gestation, representing 2 X 10(-4%) of its original wet weight. The placental thyrotropin appeared to be a 94,000-dalton protein with pI 6.0 and composed of two noncovalently associated chains of 50,000 and 44,000 daltons. The placental 94,000-dalton thyrotropin bound to TSH membrane receptors and induced specific TSH-mediated biological effects, but was structurally and immunologically distinct from TSH and hypophysical or placental gonadotropins.     

Ultracytochemical localization of guanylate cyclase in early human placenta

Previous biochemical and cytochemical studies have indicated that in human term placenta the enzyme guanylate cyclase (GC) is associated mostly with the cytosolic fraction of homogenates and localized on the syncytiotrophoblast microvillous border. In the present study we have shown cytochemically the GC particulate form in early human placenta using guanylyl-imidodiphosphate [Gpp(NH)p] as substrate and NaN3 as activator. In samples of placental villi taken from the 6th to 12th week of pregnancy, the GC reaction product was always found on the apposing Langhans cytotrophoblast and syncytiotrophoblast plasma membranes. Furthermore, GC was present on cells in mitosis of the Langhans cytotrophoblast. From the 11th week GC was also visible on basal plasma membranes of Langhans cytotrophoblast and on endothelial cells of fetal capillaries. In samples of human term placenta GC was detectable on the syncytiotrophoblast microvillous border. This suggests a shift of enzyme localization during pregnancy.     

The Oligosaccharides Produced from Sucrose by Dematium pullulans

A tetrasaccharide produced from sucrose by Dematium pullulans was obtained in a crystalline state, and its properties and physical constants were studied. By elementary analysis, molecular weight, tetrazolium test, the Raybin test, aldose value, paper chromatographic examination of partial acid hydrolyzate and enzymic hydrolyzate, periodate oxidation, and molar optical rotation, the crystalline compound was identified as β-fructofuranosyl-1-kestose trihydrate.     

Oligosaccharides isolated from Agave vera cruz

The structures of naturally occurring and enzymically synthesized oligosaccharides, consisting of fructose and glucose residues and having d.p. 3–8, in the stem of Agave vera cruz have been investigated by using methylation analysis, mass spectrometry, and p.m.r. spectroscopy. The naturally occurring trisaccharides were identified as 1-kestose and neokestose, and the tetrasaccharides as nystose and at least one other related to neokestose. The higher fractions consist of mixtures of (branched) oligosaccharides related to 1-kestose, neokestose, or 6-kestose as basic structures. The enzymically synthesized trisaccharide was identified as 1-kestose, and the tetrasaccharides as nystose. The higher fractions consist of mixtures of linear oligosaccharides related to 1-kestose and neokestose.