Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks

-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of -1,3-glucanase Lu and -1,3-glucanases with different action types—endo--1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo--1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that -1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of -1,3-glucanase from S. intermedius were more similar to those of the endo--1,3-glucanase from the marine mollusk (LIV) than exo--1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of -1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).     

The genetic mucolipidoses

Summary This review is an attempt to define a group of storage diseases which exhibit signs and symptoms of both the mucopolysaccharidoses and sphingolipidoses. Lacking some of the characteristics of the mucopolysaccharidoses while resembling to this group of thesaurismoses in other respects, these diseases frequently were described as Hurler variants.In Gm₁ gangliosidosis types I and II, Fucosidosis, Mannosidosis and in infantile Sulfatidosis with mucopolysacchariduria enzyme defects have been identified which are thought to be causally related to the diseases. In others the pathogenesis is unknown. They are tentatively named Mucolipidosis I, II and III.Supported by grants Wi 80 and Sp 63/2 of the Deutsche Forschungsgemeinschaft.     

High performance liquid chromatography of biliprotein from Bangia atropurpurea ‘Conchocelis’

Gel-filtration-purified R-phycoerythrin and phycocyanin from the filamentous phase of Bangia atropurpurea were subjected to high performance anion exchange chromatographic separation. The purified R-phycoerythrin was a mixture of three charged-isomers that were well resolved in a Bio-Gel MA7P column by elution with a NaCl gradient in phosphate buffer. These were tentatively called phycoerythrin charged-isomers and were also separable from phycocyanin and allophycocyanin by the same system. Hence, it is suggested that the ion exchange chromatographic and the sample preparation methods presented in this report can be used to distinguish the various natures of biliproteins in red algae in addition to the polyacrylamide gel electrophoresis technique. Isomers of R-phycoerythrin have the same absorption and emission spectra and were studied for their subunit compositions by a PRP-3 reverse phase column chromatography. All three charged-isomers have the same and subunits in common but differ in their subunit.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Pulse schemes for the measurement of3 JC′Cγ and3 JNCγ scalar couplings in 15N,13C uniformly labeled proteins

Pulse sequences are presented for the measurement of³J_CC and³J_NC scalar couplings for allC containing residues in¹⁵N,¹³C uniformly labeled proteins. The methodsdescribed are based on quantitative J correlation spectroscopy pioneered byBax and co-workers [Bax et al. (1994) Methods Enzymol., 239, 79–105].The combination of ³J_CC and³J_NC scalar coupling constants allows theassignment of discrete rotameric states about the ₁ torsion angle in cases where such states exist or, alternatively,facilitates the establishment of noncanonical ₁conformations or the presence of rotameric averaging. The methods areapplied to a 1.5 mM sample of staphylococcal nuclease.     

Microbial degradation of dehydrodiconiferyl alcohol,a lignin substructure model

Bacteria, yeasts, and molds which grew in a medium containing a synthetic lignin — a dehydrogenation polymer (DHP) of coniferyl alcohol — as a sole carbon source, were isolated from soil. One fungus, Fusarium solani M-13-1, was found to degrade the DHP most vigorously among the isolated organisms. It was shake-cultured in a medium containing dehydrodiconiferyl alcohol (DHCA) (I), an important lignin model compound, and the following six metabolic products were isolated and identified: 1) Phenylcoumaran--aldehydic (II) and -carboxylic compounds, 2) phenylcoumaran--aldehydic compound (IV), formed by release of a 2-carbon fragment from the phenylcoumaran--carboxylic compound, 3) 5-acetylvanillyl alcohol (V), formed by cleavage of the coumaran ring and reduction of the -aldehyde group, 4) 5-carboxyvanillyl alcohol (VI), formed by subsequent oxidation of the acetyl group, and 5) the -ether of DHCA (VII), considered to be a by-product. A degradation pathway for DHCA was proposed on the basis of these metabolic products.Non-Standard Abbreviations DHP dehydrogenation polymer - DHCA dehydrodiconiferyl alcohol - DDQ dichlorodicyano-p-benzoquinone - DDHQ dichlorodicyano-p-hydroquinone - Ar aromatic - TLC thin layer chromatography - GC-MS gas chromatography-mass spectrometry     

A two-year-old patient with an atypical expression of GM1-β-galactosidase deficiency: Biochemical,immunological, and cell genetic studies

Summary Cultured skin fibroblasts from a 2-year-old boy with an atypical form of -galactosidase deficiency have been studied. With the artificial substrate 4-methylumbelliferyl--D-galactopyranoside, 5–15% residual activity was found in fibroblasts from this patient. Most of this activity was in the monomeric A form of the enzyme, very little in the multimeric B form. Km value, pH profile, and heat lability of the mutant enzyme were similar to those of -galactosidase from control fibroblasts. Immunological studies showed that the mutant enzyme cross-reacted with an antiserum raised against human liver -galactosidase, but the catalytic activity per unit antigenic activity was lower than normal. It was demonstrated by somatic cell hybridization that the gene mutation in this patient is different from that in patients with type 1 or type 2 G_M1-gangliosidosis. No genetic complementation was found after fusion of fibroblasts from this patient with those from two other clinical variants of G_M1-gangliosidosis formerly designated type 3 and adult type 4.     

Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: Role of hydrogen peroxide

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2·–/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 M) or histamine (100 M). Superoxide dismutase (50 U/ml), which dismutates O2·minus; into H2O2 al ient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 M were used. Buffering trace amounts of iron (o-phenanthroline; 200 M) in order to inhibit úOH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca2+-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca2+-ATPases of the endoplasmatic reticulum with thapsigargin (1 M) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 M) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O· ndash; or · OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological oxidative stress associated with a progressive increase in [Ca2+]i.     

Betrachtungen zum Problem der Frostresistenzzüchtung von Tomaten

Zusammenfassung Zweijährige Selektion auf Frostresistenz bei einer Kreuzung der argentinischen Tomatensorte Platense mit der nordamerikanischen Pearl Harbour unter Freilandbedingungen erwies sich bei Überprüfung in der Kühlkammer im Vergleich zu frostempfindlichen Kultursorten als ergebnislos. Es wird daher der Schluß gezogen, daß auch bei Tomaten eine erfolgreiche Züchtung auf Frostresistenz nur durch Einkreuzung mit frostresistenten Wildarten möglich ist, die auch vorhanden sind. Unter den verfügbaren Wildarten wurde Frostresistenz beiL. peruvianum undL. hirsutum gefunden.     

Unequal specific uptake rates of α- and β-glucose by yeast andE.coli

The specific uptake rate of -glucose by yeast (S. cerevisiae) and recombinant yeast is higher than that of -glucose. On the other hand, the specific uptake rate of -glucose byE.coli and recombinantE.coli is less than that of -glucose. These results imply that the unequal specific uptake rates of optical isomers of glucose should be included in modeling and optimizing high-cell density fermentations, fed batch fermentations, and immobilized cell systems, where cell density is maintained high and glucose concentration is low.     

Human β-galactosidase and α-neuraminidase deficient mucolipidosis: Genetic complementation analysis of the neuraminidase deficiency

Summary Human -galactosidase and -neuraminidase deficient mucolipidosis [ML(gal^-neur^-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal^-neur^-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular -galactosidase and -neuraminidase levels in heterokaryons. The lack of human -galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal^-neur^-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect may occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal -galactosidase and -neuraminidase.     

Tetrads with non-reciprocal segregation in Sphaerocarpos

Summary From crosses of the mutant strains 402, 404 or 444xwild-type of Sphaerocarpos donnellii about 104000 tetrads with 4 sporelings, and about 20 000 tetrads with 3 sporelings were analyzed. No tetrad was found in which non-reciprocal segregation was induced by conversion. Therefore the frequency of conversion appears to be at least 10 times less in Sphaerocarpos than in ascomycetes. This conclusion is not contradicted by many tetrads (up to 30%), which segregated non-reciprocally from crosses of the mutant strain 429xwild-type, because these non-reciprocal segregations are completely in favor of the wild-type. In addition, plants of the phenotype wild originated in crosses of the mutant strain 429 with itself about twice as often as when crossed with the wild-type. Therefore, these findings cannot be accepted as a result of conversion in the sense of hybrid-DNA-models of crossing over. We assume that plants of the wild phenotype are brought about by the behaviour of two genetic factors.Herrn Prof. Dr. J. Straub zum 60. Geburtstag gewidmet.     

Evidence for the deficiency of β-glucosidase-activating factor in fibroblasts of patients with I-cell disease

Summary Reduced activity of -glucosidase was shown in the cultured skin fibroblasts of four patients with I-cell disease when the enzyme was tested without the use of detergents. In the presence of taurocholate and triton X100 -glucosidase activity was normal. This suggested a deficiency of a -glucosidase-activating factor in I-cell fibroblasts rather than of the enzyme itself. The deficiency of -glucosidase activity was corrected to some extent by mixing cell lysates, and more effectively by cocultivation and fusion of I-cell disease and Gaucher fibroblasts. These results present evidence for the presence of a -glucosidase-activating factor in normal and Gaucher fibroblasts. In fibroblasts of patients with I-cell disease this activator is probably deficient, as is the case for most lysosomal enzymes.     

Fine structure of sensory tubes on the antennule of Conchoecia spinirostris (Ostracoda,Crustacea)

Summary In addition to setae, the first antennae of Conchoecia spinirostris also bear soft sensory tubes (4 tubes + 1 seta; 2 tubes + 3 setae). These tubes were examined electron microscopically. Each tube is divided into 4 regions: the stem, the bulbous region, the main region, and the tip. A tube contains 40–60 multiciliated dendrites, some hypodermal cells, and nonneuronal cells, and it has a specialized cuticle. Each dendrite develops within the tube, on the terminal 5–8 m of its inner dendritic segment, approx. 25 cilia in a 9 × 2 + 0 pattern, whose rootlets are absent or only poorly developed. Each cilium splits up into 9 ramifications which extend into the tip. These ramifications partly take a spirallike course and form a ring in the distal main part beneath the cuticle. Their membranes often dilate into spindleshaped swellings. In the center of the middle and distal parts of the main region approx. 7 dendrites without cilia are located, one of them reaches into the tip. The poreless cuticle is extremely delicate and electron lucid. In contrast to the cuticle of the setae it is elastic and soft. Special substructures are described. The tubes are completely covered by a filamentous surface coat. Because of the structure and the thin walled nature of the cuticle, permeability for dissolved substances is assumed. The ciliary ramifications are likely to represent the receptive apparatus. The sensory tubes are interpreted as chemoreceptors. They can best be compared with the chemoreceptors of certain crustaceans, but differ strongly from the types of sensilla found in insects.Supported by project 3540 of the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich. The author is deeply indebted to Prof. Gamulin (Dubrovnik) for his support and to Prof. R. Riedl and Dr. W. Klepal for helpful discussions     

Secretion of unphosphorylated and phosphorylated xyloside-induced glycosaminoglycan chains

Secretion ofp-nitrophenyl--xyloside-induced glycosaminoglycan chains in cultured fibroblasts is considered to involve transport through the endoplasmic reticulum and the Golgi apparatus. Purified glycosaminoglycans from fibroblast secretions contain small amounts of covalently bound [³²P]phosphate. However, exhaustive digestion with chondroitin AC and ABC lyases yields an unphosphorylated linkage region tetrasaccharide in the majority of all polysaccharide chains. The phosphate label is associated predominantly with material of the expected behavior of linkage region hexasaccharides. Thus, phosphorylation is not a prerequisite to secretion of xyloside-induced glycosaminoglycan chains.     

Role of the glycosaminoglycan microenvironment of hyaluronidase in regulation of its endoglycosidase activity

The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the 1-3 and 1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking—(1e-4e) and (1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.     

Immobilization of digitoxin 12β-hydroxylase,a cytochrome P-450-dependent enzyme from cell cultures of Digitalis lanata EHRH

Attempts were made to immobilize digitoxin 12-hydroxylase, a membrane-bound, cytochrome P-450-dependent monooxygenase from cell cultures of Digitalis lanata. The optimum procedure was the entrapment of microsomes in 2% alginate by crosslinking the polysaccharide chains with CaCl₂. After the immobilization of the enzyme about 70% of its activity was retained. The kinetic data such as the pH optimum and the optimum substrate concentrations were identical for the immobilized enzyme and freely suspended microsomes. Using -methyldigitoxin as a substrate enzyme activity could be observed for more than 20 h. A continuous flow system for immobilized digitoxin 12-hydroxylase is described.Abbreviations -mdg -methyldigoxin - -mdt -methyldigitoxin     

Kinetic properties of β-glucosidase from Aspergillus ornatus

Summary Kinetic properties of extracellular -glucosidase from Aspergillus ornatus were determined. The pH and temperature optima for the enzyme were found to be 4.6 and 60°C, respectively. Under these conditions, the enzyme exhibited a K _m (p-nitrophenyl--glucoside) value of 0.76±0.11 mM. The activation energy for the enzyme was 11.8 kcal/mol. Several divalent metal ions inhibited -glucosidase activity, some of which showed inhibition of enzyme activity only at higher concentrations. Ag²⁺ was the most potent inhibitor. A metal chelating agent, EDTA, also inhibited -glucosidase activity. Except for trehalose, glucose, glucono--lactone, cellobiose, gentiobiose, laminaribiose, maltose and isomaltose inhibited -glucosidase activity. Glucose was found to be a competitive inhibitor, whereas glucono--lactone and other -linked disaccharides were noncompetitive (mixed) inhibitors of the enzyme.     

Analysis of a case of mutagen specificity in Neurospora crassa. 3. Fractionated treatment with diepoxybutane (DEB)

Summary Weak doses of DEB given before or after a moderately high dose act as booster for the production of adenine-reversions. Fractionation of a moderate or high dose into a succession of weak ones yields a dose-effect curve that lies above the linear curve expected for additivity of the fractions but below that found after continuous exposure. The results lend support to the view that DEB, in addition to producing potential adenine-reversions in DNA, promotes their realization by its effect on some cellular process or processes, and that this is the cause of the steep dose-response to continuous exposure.