Ultrastructural study of skin and eye of UV‐B irradiated ayu Plecoglossus altivelis

A scanning electron microscopic study of the skin and eye of UV‐B radiated ayu Plecoglossus altivelis (age 30 days, mean ±  s . e . total length: 16·25 ± 0·11 mm) under laboratory condition showed marked changes when compared with the control fish without UV‐B radiation. The exposure of the fish to the radiation resulted in the destruction of microridges in the epidermis and exposed neuromast cells of the skin. Domed protrusions were also more common in the skin of UV‐B radiated fish than in the control fish. The appearance of mucus in both groups was different. In the control skin the mucus was spread over a wide area whereas in the treated fish the mucus was concentrated in a small area. The anastomozing structures of the microridges of the eyes were lost in UV‐B radiated fish and the microridges themselves were fewer in number, fragmented, and aggregated. Mucus cells, prominently visible in the control fish, were distorted in the treated fish. Cell contours were irregular in UV‐B radiated fish and cell to cell contacts had been lost in this group.     

Glycolipids Isolated from Aplysia kurodaiCan Activate Cyclic Adenosine 3', 5'-Monophosphate-Dependent Protein Kinase from Rat Brain

Abstract: Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonogly-cosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGalβ 1→3GalNAcα 1→3 [6'- O -(2-aminoethylphosphonyl) Galα1→2] (2-aminoethylphosphonyl→6) Glcβ 1→4GICβ1→1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 μ M. Activation of cAMP-kinase was maximal with 250 μ M SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Two types of rat gastric mucus glycoprotein subunits

Gastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two "subunits" (Mw 4.4 x 10(5) and 6 x 10(6)) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4 x 10(5). This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4 x 10(5)) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.     

Bovine Pituitary Membrane Glycoproteins Contain β-N-Acetylgalactosaminylated N-Linked Sugar Chains

Abstract: Bovine pituitary glycoprotein hormones contain unique N-linked sugar chains with GalNAcβ1 → 4GlcNAc structure in their outer chain moieties. In the present study, whether bovine pituitary membrane glycoproteins contain the sugar chains with the disaccharide structure was investigated. Western blot analysis of the membrane glycoproteins using Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with β- N -acetylgalactosamine residue(s), showed that most protein bands detected with Coomassie Brilliant Blue staining bind to WFA. However, no WFA binding was observed for the bands after treatment of the blotted filter with jack bean β- N -acetylhexosaminidase or N -Glycanase. The WFA-positive bands were also detected in membrane glycoprotein samples from bovine cerebrum, cerebellum, and medulla oblongata, although their expression levels were low. Structural analysis of the oligosaccharides released by hydrazinolysis from the pituitary membrane glycoproteins by serial lectin column chromatography and sequential exoglycosidase digestion revealed that the major oligosaccharides, which bound to a WFA-agarose column, are of biantennary complex type with one and two GalNAcβ1 → 4GlcNAc groups in their outer chain moieties. These results indicate that the β- N -acetylgalactosaminylation is not unique to the glycoprotein hormones but occurs to most bovine pituitary glycoproteins.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Lipid content and fatty acid composition of target tissues in wild Perca fluviatilis females in relation to hepatic status and gonad maturation

Variation in liver ultra‐structure and composition in relation to energy mobilization was investigated in female perch Perca fluviatilis from the Meuse River between August 2001 and June 2002. In April, just before spawning, the lipo‐somatic index ( I _F) was 0·3%, the gonado‐somatic index ( I _G) was 28% and the total lipid content of the liver was 2·53%. The average areas of lipid droplets and mitochondria were 0·05 and 0·06 μm², respectively. Glycogen supply reached 7·9% of the total area of the hepatocyte. During the sexual resting period, females accumulated energy in perivisceral fat and in the liver to reach 1·6% I _F and 4·85% of liver lipid content in August with lipid droplets average size of 0·09 μm² and glycogen average area of 15%. Liver cells contained a weakly developed rough endoplasmic reticulum (RER) and a great number of small mitochondria (average size 0·02 μm²). The I _G was 0·6% at this time. During the whole annual cycle, the average lipid content of female liver never exceeded 3·9 ± 1·9%. The concentration of docosahexaenoic (DHA), linolenic and linoleic acids increased in mature gonads while linolenic and linoleic acids decreased in the liver during the same period. Fatty acid composition of muscles of perch was characterized by a high content of DHA.     

Histidine carboxylase of Leuconostoc œnos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene

Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc ' nos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively α and β. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. ' nos 9204 HDC was synthesized as a precursor proHDC π₆ (Mr 205 000). A cleavage between Ser-81 and Ser-82 generated the α (Mr 25 380) and β (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (αβ)₆. At the optimal pH of 4·8, the HDC activity exhibited a simple Michaelis-Menten kinetic ( K _m = 0·33 mmol l⁻¹, V _max = 17·8 μmol CO₂ min⁻¹ mg⁻¹), while at pH 7·6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor ( K _i = 32 mmol l⁻¹). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.     

Production of mucin degrading sulphatase and glycosidases by Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron NCTC 10582 grown in media containing pig gastric mucin was found to be capable of producing all the glycosidases required to degrade the carbohydrate moieties of human colonic mucin. These are α-fucosidase, β -galactosidase, α- N -acetylgalactosaminidase, β-N -acetylglucosaminidase and neuraminidase. Moreover, a novel glycosulphatase was identified using glucose-6-sulphate as substrate. This enzyme has a Km of 43·4 mmol/l and a pH optimum of 5·0. The bacteria, when cultured for 24 h in broth, were capable of removing 18% of [³⁵S]-sulphate from [³⁵S]-labelled mucin and of removing 15% of [³H]-glucosamine from [³H]-glucosamine-labelled human colonic mucin. The results suggest that this bacterium is likely to play an important role in mucus degradation in the human colon.     

A new technique to gather 3‐D spatial information using a single camera

A new technique using a single camera and shadows to determine 3‐D spatial positions of fishes in the laboratory is described. The apparatus consisted of a large aquarium (2·0 × 1·5 × 0·4 m), a wide‐angle camera mounted above and two light sources to cast shadows to either side of the fish. Using image analysis and vector mathematics, aquarium objects were plotted within 1·5 cm of their actual location along the x ‐, y ‐ and z ‐axis. The technique was also successful in quantifying changes in 3‐D spatial pattern of juvenile fish, Atlantic cod Gadus morhua (7·4–8·6 cm standard length, L _S) and cohabitant piscivorous shorthorned sculpin Myoxocephalus scorpinus (12·0–25·8 cm L _S), at these same viewing fields. The new technique should have a wide application, largely because it is potentially less expensive, laborious and invasive than alternative methods for determining 3‐D positions of fishes.     

The enzymatic reaction for the production of panose and isomaltose by glucosyltransferase from Aureobasidium

Glucosyltransferase from Aureobasidium produced 212 mg ml^-1 of glucosyl-oligosaccharides (panose: Glcα1→6Glcα1→4Glc 189 mg ml^-1 and isomaltose: Glcα1→6Glc 23 mg ml^-1) from maltose: Glcα1→4Glc at a high concentration (500 mg ml^-1) and the efficiency of production was 42-4%. The enzymatic reaction from maltose to panose is reversible but that from panose to isomaltose is not.     

The Uptake and Utilization of Bacteria, Amino Acids and Carbohydrate by the Rumen Ciliate Entodinium longinucleatum in Relation to the Sources of Amino Acids for Protein Synthesis

Entodinium longinucleatum grown in vitro in the presence of bacteria engulfed a wide range of bacterial species at rates of 130–3400 bacteria/h/protozoon (from suspensions of 10 bacteria/ml), but showed a preference for Klebsiella aerogenes and Proteus mirabilis which occurred in the growth medium. Some of the bacteria were digested with release of soluble material into the medium. Free amino acids were incorporated by the protozoa in the presence of chloramphenicol at rates of 5·4–15·1 nmol/h/10⁶ protozoa and approximately 40% of the amino acid-carbon was incorporated into protein. There was no appreciable synthesis of protozoal protein from carbohydrate. Evidence was obtained that the protozoa obtained the amino acids required for growth largely from engulfed bacteria.     

Glycoconjugate distribution in gastric fundic mucosa of Umbrina cirrosa L. revealed by lectin histochemistry

The stomach of adult shi drum Umbrina cirrosa was investigated using a battery of nine horseradish peroxidase‐conjugated lectins combined with enzymatic treatment, in order to distinguish glycoconjugate sugar residues. Epithelial cells showed the presence of galactosyl(β1→4)N‐acetylglucosamine, mannose, N‐acetylgalactosamine, N‐acetylglucosamine, fucose and sialic acid‐galactosyl(β1→3)N‐acetylgalactosamine residues. Gastric pits had similar sugar residues with the exception of N‐acetylgalactosamine which was less diffused. Gastric glands were characterized by the presence of glycoconjugates containing galactosyl(β1→3)N‐acetylgalactosamine, N‐acetylglucosamine, galactosyl(β1→4) N‐acetylglucosamine, N‐acetylgalactosamine and a small amount of sialic acid linked to N‐acetylgalactosamine.     

Phosphonoglycoprotein from Metridium senile--heterogeneity of glycoproteins containing aminoethylphosphonic acid.

1. After separation by SDS gel-chromatography, analysis of AEP-containing glycoproteins from M. senile, indicated 66% amino acids with 220 AEP res./1000 res. and 30% carbohydrate for high mol. wt (greater than 10(7) forms and 80% amino acids with 25-50 AEP res./1000 res. and 10% carbohydrate for low mol. wt (2-4 x 10(4) forms. 2. Uronic acids, sulfate, lipid, and sialic acids were absent. 3. Mild base digestion released AEP-hexosamine containing oligosaccharides and destroyed ser-thr residues in the high mol. wt components. 4. Phosphonoglycoproteins appear to be acidic connective tissue components with AEP linked to hexosamine containing oligosaccharide side chains.     

Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β- d -Gal p NAc-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p NAc-(1→4)-β- d -Gal p -(1→3)-α- d -Gal p NAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution of d -GlcNAc for d -Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 ( d -GlcNAc) and O117 ( d -Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.     

Purification and Characterization of Glycoprotein from the Skin Mucus of the Rainbow Trout,Salmo gaivdneri

Mucus glycoprotein (RGP) was purified and characterized from the skin mucus of rainbow trout, Salmo gairdneri. RGP was found to contain 30.1% NeuAc, 26.0% GalNAc, 5.0% Gal, and 26.0% amino acids. The protein moiety of RGP is very rich in Thr (32.4 mol%). Neither NeuGc nor KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) was found in RGP. Alkaline borohydride treatment of RGP yielded a major disaccharide alditol, NeuAcα2→6GalNAc-ol and more than 4 minor oligosaccharide alditols including NeuAc→(GalNAcα1→)GalNAc-ol. It was evident that an average RGP molecule has approximately 500 NeuAc-containing oligosaccharide chains, which are attached to the Thr and Ser residues of the protein moiety and spaced at an average of 3 amino acids apart.     

LIPID COMPOSITION OF OPTIC NERVE MYELIN

Abstract— Myelin was isolated from bovine optic nerves by differential ultracentrifugation and its lipid composition was analysed. Optic nerve myelin contained 76·3 per cent lipid. The major lipids were cholesterol, ethanolamine glycerophosphatides (EGP) and cerebroside. Serine glycerophosphatides (SGP), sphingomyelin and cerebroside sulphate were present in smaller proportions. EGP and SGP contained 34·6 and 0·5 per cent aldehydes. The major fatty aldehydes were palmitaldehyde, stearaldehyde and octadecenaldehyde. The fatty acids of EGP, SGP and choline glycerophosphatides (CGP) were chiefly 16:0, 18:0 and 18:1, with small proportions of 20 and 22 carbon polyunsaturates. The sphingolipids contained predominantly saturated and monounsaturated fatty acids of chain lengths of 20–26 carbon atoms. Optic nerve myelin and white matter myelin resembled one another closely in overall lipid composition and in the fatty acid compositions of their constituent lipids. Optic nerve myelin and white matter myelin are chemically similar membranes, but both of these differ in their lipid composition from spinal root myelin.     

Morphological differentiation and resource polymorphism in three sympatric whitefish Coregonus lavaretus(L.) forms in a subarctic lake

Three field‐identified whitefish Coregonus lavaretus forms in Lake Muddusjärvi, Finland, were compared in morphology, diet and prey size. First, these forms were studied with univariate and multivariate analysis to assess morphological divergence at a higher resolution level than in the field. Second, stomach contents were analysed to estimate diet‐overlap among forms. Finally, the relationship between prey size and morphology was examined. The whitefish were assigned to the initial field‐classification with 99·2% and 98·8% accuracy for morphologic and meristic traits, respectively. The small sparsely‐rakered form (SSR) had the shortest rakers and largest gillraker space, followed by the large sparsely‐rakered form (LSR) with intermediate gillraker length and gillraker space, while densely‐rakered whitefish (DR) had the longest rakers and smallest gillraker space. The two sparsely‐rakered whitefish forms (LSR and SSR), consumed mainly benthic macroinvertebrates, while densely‐rakered whitefish (DR), utilized pelagic food items. Average diet‐overlaps between whitefish forms were low in June‐September (Schoener's α = 0·02 − 0·23). Gillraker number and length were negatively correlated to prey length in the diet ( r  = −0·73, and r  = −0·60), while gillraker space was positively correlated with prey length ( r  = 0·81). The fact that these whitefish forms were morphologically and ecologically segregated, and that gillraker traits probably have a functional value in food selection, further suggests that natural selection has been important in structuring life‐history trajectories into divergent niche use.