Production of 4-methylumbelliferyl heptanoate hydrolase by Escherichia coli exposed to seawater.

The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied. In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days. Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation.     

Production of 4-methylumbelliferyl heptanoate hydrolase by Escherichia coli exposed to seawater

The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied. In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days. Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation.     

Chitosanase-catalyzed hydrolysis of 4-methylumbelliferyl beta-chitotrioside.

4-Methylumbelliferyl beta-chitotrioside [(GlcN)(3)-UMB] was prepared from 4-methylumbelliferyl tri-N-acetyl-beta-chitotrioside [(GlcNAc)(3)-UMB] using chitin deacetylase from Colletotrichum lindemuthianum, and hydrolyzed by chitosanase from Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)(3)-UMB was confirmed by (1)H-NMR spectroscopy and mass spectrometry. When the (GlcN)(3)-UMB obtained was incubated with chitosanase, the fluorescence intensity at 450 nm obtained by excitation at 360 nm was found to increase with proportion to the reaction time. The rate of increase in the fluorescence intensity was proportional to the enzyme concentration. This indicates that chitosanase hydrolyzes the glycosidic linkage between a GlcN residue and UMB moiety releasing the fluorescent UMB molecule. Since (GlcN)(3) itself cannot be hydrolyzed by the chitosanase, (GlcN)(3)-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. The k(cat) and K(m) values obtained for the substrate (GlcN)(3)-UMB were determined to be 8.1 x 10(-5) s(-1) and 201 microM, respectively. From TLC analysis of the reaction products, the chitosanase was found to hydrolyze not only the linkages between a GlcN residue and UMB moiety, but also the linkages between GlcN residues. Nevertheless, the high sensitivity of the fluorescence detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.     

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin.

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.     

Synthesis and stability assay of 4-methylumbelliferyl (1-->3)-beta-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 3 are described. The (1 --> 3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 --> 3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 --> 3)-beta-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 --> 3)-beta-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl4) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with beta-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

Metabolism of ketone bodies, oleate and glucose in lymphocytes of the rat.

Isolated incubated lymphocytes utilized acetoacetate, 3-hydroxybutyrate or oleate at about 0.5 mumol/min per g dry wt. These rates were not markedly affected by concanavalin A or by starvation of the donor animal. When ketone bodies replaced glucose in the culture medium, they could not support lymphocyte proliferation when cells were cultured for 48 h. Addition of oleate (0.5 mM) to isolated lymphocytes increased the rate of O2 consumption markedly, suggesting that it could contribute about 30% to O2 consumption. The rate of oleate uptake and the stimulated rate of O2 consumption were maximal at 0.5 M-oleate; this is in contrast with the effect in some other tissues, in which the rate of fatty acid oxidation is linear with concentration up to about 2 mM. Since the normal plasma concentration of fatty acid in the fed state is about 0.5 mM, this suggests that lymphocytes can utilize fatty acids at a maximal rate in the fed state. Ketone bodies or oleate decreased the rate of glucose utilization by incubated lymphocytes; ketone bodies decreased the rate of pyruvate oxidation and increased the intracellular concentration of hexose monophosphate and citrate, suggesting that 6-phosphofructokinase is inhibited by citrate, and hexokinase by glucose 6-phosphate. These effects may be important not so much in conserving glucose in the whole animal but in maintaining the concentrations of glycolytic intermediates necessary for biosynthetic processes during proliferation.     

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets.

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with bis-(p-nitrophenyl) phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards 5'-dTMP-P-nitrophenyl ester, which is probably associated with intracellular membrane structures in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.     

Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.

A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.     

Parameter comparison of white matter diffusion tensor imaging (DTI) in rhesus macaques (Macaca mulatta)

In this study, we analyzed diffusion tensor imaging(DTI) results of brain white matter in rhesus macaques(Macaca mulatta) with four different parameter settings and found that the sequence A(b=1 000 s/mm2, spatial resolution=1.25 mm×1.25 mm× 1.25 mm, numbers of direction=33, NSA=3) and B(b=800 s/mm2, spatial resolution=1.25 mm×1.25 mm×1.25 mm, numbers of direction=33, NSA=3) could accurately track coarse fibers. The fractional anisotropy(FA) derived from sequence C(b=1 000s/mm2, spatial resolution=0.55 mm×0.55 mm×2.5 mm, direction number=33, NSA=3) was too fuzzy to be used in tracking white matter fibers. By comparison, the high resolution and the FA with high contrast of gray matter and white matter derived from sequence D(b=800 s/mm2, spatial resolution=1.0 mm×1.0 mm ×1.0 mm, numbers of direction=33, NSA=3) qualified in its application in tracking both thick and thin fibers, making it an optimal DTI setting for rhesus macaques.     

Protein-sugar interactions: Environmental effect on the fluorescence of O-(4-methylumbelliferyl)-glycosides

We have investigated the effect of 12 solvents and several amino acids on the fluorescence of O-(4-methylumbelliferyl)-glycosides. We showed that: i) the fluorescence quenching is not related to the dielectric constant of the solvents: the fluorescence intensity was maximal in water (d=80) and in acetic acid (d=6.2) and was at least ten times lower in acetone (d=21) and in dioxane (d=2.2); ii) the fluorescence of O-(4-methylumbelliferyl)-N-acetyl-β-glucosaminide is not quenched in the presence of various amino acids including arginine, asparagine, aspartate, histidine, leucine, phenylalanine and proline; iii) the fluorescence of O-(4-methylumbelliferyl)-glycoside is quenched by sulfur, phenol and indole amino acids or derivatives containing sulfur, phenol or indole groups. The changes in fluorescence intensities of O-(4-methylumbelliferyl)-glycosides upon binding to concanavalin A, wheat germ agglutinin and lysozyme are discussed with regard to the amino acid content of their binding sites.     

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with $\text{bis-(}\text{p-}\text{nitrophenyl)}$ phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards $\text{5′ -}\text{dTMP}\text{-p-}\text{nitrophenyl}$ ester, which is probably associated with intracellular membrane structure in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

Invaginations in plant cell protoplasts containing mycoplasma-like bodies (MLO)

MLO containing invaginations were found in protoplasts of phloem parenchyma cells in symptomless young leaves ofRibes houghtonianum Jancz. infected with a yellows disease. The invaginations originate between the cell wall and plasmalemma, usually at plasmodesmata, and change apparently into superficial vesicles in the protoplast; they are entirely or partially limited by host plasmalemma. The formations mentioned occur in parenchyma cells which contain normal organelles. Sometimes they are divided by a smooth membrane system enclosing MLO. Besides MLO the invaginations contain in some cases slimy fibrils resembling the P-protein in sieve tubes. The MLO bodies seen in invaginations have usually a diameter of 50–250 nm and their plasmalemma (unit membrane) is identical with the plasmalemma of MLO bodies occurring in sieve tubes. However, only few MLO bodies in invaginations are electron dense, so that they resemble naturally degenerated forms of MLO. Similar MLO containing invaginations were formerly described from some leafhoppers transmitting MLO.     

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).     

Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system

Four monoclonal antibodies are characterized that have been obtained from a fusion of mouse myeloma P3-NS1/1-Ag4-1 with spleen cells from BALB/c mice immunized with white matter from bovine corpus callosum. The corresponding antigens (O antigens) are designated O1, O2, O3, and O4. The localization of these antigens was investigated by indirect immunofluorescence in cultures of early postnatal mouse cerebellum, cerebrum, spinal cord, optic nerve, and retina. When tested on live cultures none of the O antibodies reacted with the surface of astrocytes, neurons, or fibroblasts, however, all are positive on the surface of oligodendrocytes. The identity of these cells was determined by double-immunolabeling experiments with indpendent cell-type-specific antigenic markers (glial fibrillary acidic protein, tetanus toxin receptors, fibronectin, and galactocerebroside). Antigen O1 is exclusively expressed on galactocerebroside-positive cells, whereas O2, O3, and O4 are expressed on additional cells that are negative for any of the markers tested. None of the O antigens is expressed on the surface of cultured retinal cells. In fresh-frozen sections of adult mouse cerebellum all O antigens are detectable in white matter tracts and in vesicular structures of the granular layer. O2 and O3 antigens are in addition detectable in GFA protein-positive radial fibers in the molecular layer. In fixed cerebellar cultures, where intracellular antigens are accessible, O1, O2, and O3 antibodies label astrocytes in a GFA protein-like pattern. O antigens are expressed in mouse, rat, chicken, and human central nervous systems. O antibodies belong to the IgM immunoglobulin subclass and have been used in complement-dependent cytotoxic elimination of cerebellar oligodendrocytes in culture. At limiting antibody dilutions all processes of oligodendrocytes are preferably lysed over cell bodies.