Glycosidases and Cerebellar Ontogenesis in the Rat

Abstract: Five glysosidases (α- and β-D-mannosidase, β-D-galactosidase, β- N -acetyl-glucosaminidase, and α-L-fucosidase) were studied during the postnatal development of the rat cerebellum. Each glycosidase has a particular developmental pattern. Transient decreases in the specific activities of β-mannosidase, β-D-galactosidase, and β-D- N -acetyl-glucosaminidase could be correlated with the phase of massive cell multiplication. A possible more specific role for α-D-mannosidase is discussed.     

Distribution of O-glycosylhydrolases in marine fungi of the Sea of Japan and the Sea of Okhotsk: Characterization of exocellular N-acetyl-β-D-glucosaminidase of the marine fungus <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The capacity to produce exocellular enzymes was studied for 92 samples of fungi from various marine habitats in the Sea of Okhotsk (78 strains) and the Sea of Japan (14 strains). Strains producing highly active glycanases and glycosidases were found. Synthesis of O-glycosylhydrolases was stimulated by addition of laminaran to the nutrient medium. Highly purified N-acetyl-β-D-glucosaminidase was isolated from the marine fungus Penicillium canescens. The molecular weight of the enzyme determined by SDS-Na-electrophoresis was 68 kDa. The enzyme displayed maximum activity at pH 4.5 and temperature 45°C. Inactivation half-time of the enzyme at 50°C was 25 min. N-acetyl-β-D-glucosaminidase hydrolyzed both β-glucosaminide and β-galactosaminide bonds and possessed a high transglycosylating activity.     

The binding requirements of monkey brain lysosomal enzymes to their immobilised receptor protein

The lysosomal enzyme binding protein (receptor protein) isolated from monkey brain was immobilised on Sepharose 4B and used to study the binding of brain lysosomal enzymes. The immobilised protein could bind \-D-glucosaminidase, α-D-mannosidase, α-L-fucosidase and2-D-glucuronidase. The bound enzymes could be eluted either at an acid pH of 4.5 or by mannose 6-phosphate but not by a number of other sugars tested. Binding could be abolished by prior treatment of the lysosomal enzymes with sodium periodate. Alkaline phosphatase treatment of the enzymes did not prevent the binding of the lysosomal enzymes to the column but decreased their affinity, as seen by a shift in their elution profile, when a gradient elution with mannose 6-phosphate was employed. These results suggested that an ‘uncovered’ phosphate on the carbohydrate moiety of the enzymes was not essential for binding but can enhance the binding affinity.     

Enzyme production by lactobacilli and the potential link with infective endocarditis

H.J. OAKEY, D.W.S. HARTY AND K.W. KNOX. 1995. Fifty-six strains of lactobacilli were examined for the production of glycosidases and proteases (arylamidases) that could be associated with the ability to grow in vivo and/or be a factor in the pathogenesis of endocarditis. The strains were from seven species, with an emphasis on Lactobacillus rhamnosus and Lact. paracasei subsp. paracasei , both of which have been associated with endocarditis and provided 12 of the 13 strains isolated from cases of the disease. Other species were Lact. acidophilus, Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris.
Commonly expressed glycosidase activities were α-D-galactosidase and β- N -acetyl-D-glucosaminidase followed by β-D-glucosidase and α-L-fucosidase. The combined production of β- N -acetyl-D-glucosaminidase and α-D-galactosidase was a feature of the endocarditis isolates. In contrast, β-D-galactosidase was produced by very few of the strains within species implicated in endocarditis but most of the strains of Lact. salivarius, Lact. fermentum and Lact. oris.
The most commonly produced arylamidases active against substrates employed for testing human blood clotting cascade were activated protein C(Ca)-like, activated factor X(Xa)-like and Hageman factor-like followed by kallikrein-like and chymotrypsin-like enzymes. Kallikrein-like enzyme activity was shown more frequently by strains from species commonly isolated from cases of endocarditis ( Lact. rhamnosus and Lact. paracasei subsp. paracasei ) than from other oral species ( Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris ).
The data indicate that some lactobacilli can produce enzymes that would enable the breakdown of human glycoproteins and the synthesis and lysis of human fibrin clots, characteristics which aid the colonization and survival of bacteria infecting an endocarditis vegetation.     

b-D-glucosidase activity in pelargonium and poinsettia leaves infected by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers. ex Fr.

The involvement of β-D-glucosidase activity in grey mould was studied in two ornamental plant species attacked by Botrytis cinerea. β-D-glucosidase activity in the susceptible pelargonium cultivar ‘Shiva’ gradually increased with the disease development in the leaf spots and their surroundings. The endogenic level of the studied hydrolase in the resistant pelargonium ‘Cascade’ was several times higher than in the susceptible cultivar ‘Shiva’ and in principle underwent no changes after inoculation. The postinfection increase in the activity of β-D-glucosidase noted in the leaves of the susceptible poinsettia cultivar ‘Malibu Red’ was evidently weaker in the intensity, but its tendencies were similar to those of the susceptible pelargonium cultivar. In the leaves of the medium-resistant poinsettia ‘Coco White’ the constitutional level of β-D-glucosidase was 2-3-fold higher in that cultivar than in the susceptible cv. ‘Malibu Red’. In attacked leaves of ‘Coco White’, the enzyme activity continued to increase temporarily until the 3^rd h after inoculation. The process of healthy leaf senescence in both species had no significant influence on the change of the studied enzyme activity which was generally low. A high activity of β-D-glucosidase was also observed in the homogenate prepared from mycelium and in the fungal spores.     

O-glycosylhydrolases of embryos of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> and effect of some natural substances on their biosynthesis

Embryos of the sea urchin Strongylocentrotus intermedius have been found to contain o-glycosyl hydrolases: highly active 1,3-β-D-glucanase and β-D-mannosidase as well as a lower activity of β-D-glucosidase and β-D-galactosidase. Dynamics of changes of the enzyme activities has been studied at various stages of the sea urchin development. There has also been studied effects of some substances (natural fucoidans, β-1.3; 1.6-glucans formed by enzymatic synthesis as well as protein inhibitor of marine mollusc endo-1,3-β-D-glucanases) on development of the embryos and biosynthesis of 1,3-β-D-glucanase and α-D-mannosidase.     

Screening for glycosidase activities of lactic acid bacteria as a biotechnological tool in oenology

The aim of this study was to evaluate the ability from a number of lactic acid bacteria isolated from different sources to produce glycosidase enzymes. Representative isolates (225) from clusters obtained after genotyping, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis, of 1,464 isolates, were screened for β-D-glucosidase activity. Thirty-five of them were selected for subsequent analysis. These strains were able to hydrolyze α-D-glucopyranoside, β-D-xylopyranoside and α-L-arabinofuranoside although β-D-glucosidase activity was the predominant activity for 22 of the selected strains. Only some of them did so with α-L-rhamnopyranoside. All of these were from wine samples and were identified as belonging to the Oenococcus oeni species using Amplification and Restriction Analysis of 16S-rRNA gene (16S-ARDRA). When the influence of pH, temperature and ethanol or sugars content on β-D-glucosidase activity was assayed, a strain-dependent response was observed. The β-D-glucosidase activity occurred in both whole and sonicated cells but not in the supernatants from cultures or obtained after cell sonication. Strains 10, 17, 21, and 23 retained the most β-D-glucosidase activity when they were assayed at the conditions of temperature, pH, ethanol and sugar content used in winemaking. These results suggest that these strains could be used as a source of glycosidase enzymes for use in winemaking.     

Embryonic development of an endoparasitoid,Microplitis croceipes (Hymenoptera: Braconidae) in cell line-conditioned media

Summary Embryos of the parasitoidMicropolitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived from fat body from an atypical hostLymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace’s, and ExCell 400. The developmental response ofM. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace’s media promoted development to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ hand stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace’s media had a significant effect on eggs attaining germ band stage compared with the Grace’s control medium. However, Grace’s medium conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly higher percentage of hatching eggs than the Grace’s control medium. Although the BCIRL-HZ-AMI cell line, which is derived from the parasitoid’s typical host, did not induce hatch in either IPL-52B medium or Grace’s medium, it promoted hatch in TC-199 and Excell 400 media. Fat body taken from the same species that the cell lines were derived from was a better predictor of a cell line’s embryotrophic activity in Grace’s medium rather than in IPL-52B medium. Thus, the composition of the medium and the species and tissue type of the cell line source must be evaluated interactively to determine optimal conditions for promoting development ofM. croceipes in vitro.     

I-Cell disease (mucolipidosis type II) serum hydrolases in obligate heterozygotes

Summary The mean activities of N-acetyl-β-D-glucosaminidase, β-D-glucuronidase, arylsulphatase A and β-D-galactosidase in the serum of 10 proved heterozygotes for the mutant gene causing I-cell disease (ICD) are significantly different from those in age-matched control sera. Overlapping of individual results in both groups renders assay of serum acid hydrolases an impractical method of reliable detection of the ICD heterozygous genotype. That the mutant gene is also partially expressed in heterozygote serum, may be useful in assessing existing hypoteses on the nature of its primary metabolic defect.

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Zusammenfassung Es zeigte sich, da? die durchschnittliche Aktivit?t der N-Acetyl-β-D-Glucosaminidase, der β-D-Glucuronidase, der Arylsulfatase A und der β-D-Galaktosidase im Serum von 10 Patienten, die gesichert heterozygot für das Gen waren, das die Inclusion-Cell Disease (ICD) verursacht, sich signifikant unterscheidet von der Aktivit?t in Seren einer altersentsprechenden Kontrollgruppe. Da sich die beiden Gruppen hinsichtlich ihrer Aktivit?ten der sauren Serumhydrolasen überschneiden, erscheint ein Bestimmen dieses Enzyms für eine wohlfundierte Untersuchung auf einen heterozygoten Genotypus bezüglich der ICD ungeeignet. Die Tatsache, da\ sich im Serum eines Heterozygoten das mutierte Allel ebenfalls teilweise manifestiert, mag als eine Hilfe gelten für die Entscheidung, welche von den z. Z. bestehenden Hypothesen bezüglich der Ursache dieses prim?r metabolischen Defektes die richtige ist.

     

Axial Distribution of Glycosidases in Relation to Cellular Growth and Ageing in Pisum sativum Root

Tanimoto, E. 1985. Axial distribution of glycosidases in relationto cellular growth and ageing in Pisum sativum root.—J.exp. Bot. 36: 1267–1274. Eight glycosidase activities were measured in relation to cellulargrowth and ageing along the axis of pea roots. The highest activities of ß-D-fucosidase and ß-D-glucosidasewere in the apical 1.0 mm segment containing the root cap andmeristem. Activities of -L-arabinosidase, -D-mannosidase, ß-D-galactosidase, -D-glucosidase and ß-D-xylosidase werehighest in the segment between 1 and 2 mm from the tip and containingyoung elongating cells with high growth potential. The activityof -D-galactosidase was high between 1 and 2 mm from the roottip, decreasing to a minimum 4-5 mm from the tip and increasingagain up to the base of root. This distribution of enzyme activitiesrelative to the root tip remained unchanged during 28 h whilethe root length doubled. No -L--fucosidase, ß-L-fucosidaseand -D-xylosidase activities were detected. Key words: Pisum sativum, root growth, glycosidase, galactosidase, mannosidase     

Ref cell hydrolases: Glycosidase activities in human erythrocyte plasma membranes

Summary Human erythrocyte plasma membranes were found to contain the following glycosidases: α- and β-glucosidase, α- and β-galactosidase, α- and β-fucosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, β-xylosidase and α-mannosidase. All the enzymes except β-fucosidase had activity interpreted to be on the external surface of the plasma membrane. The enzymes had optimum pH values of 5.2 to 5.0 and temperatures of 37 to 40°C. The enzymes were not greatly activated by divalent cations but Hg⁺⁺ and Pb⁺⁺ were inhibitory. The enzyme extract of the human erythrocyte plasma membranes liberated carbohydrate from intact red cells, which lead to the speculation that the glycosidases might function to modify the erythrocyte plasma membrane. The author is a Research Career Development Awardee of the National Institute of General Medical Sciences.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

Protein kinase CK2 is a serine/threonine kinase expressed in organisms from yeast to human and is composed of a catalytic subunit (α or α’) and a regulatory subunit (β) forming a holoenzyme with the possible subunit combinations α₂β₂, α’₂β₂, or αα’β₂. This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2α’ and CK2β subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2α’ and CK2β are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2α’ and CK2β by immunohistochemistry suggests that CK2α’ and CK2β are localized in the nuclei of Sertoli cells in 5-day-old rats, whereas they appear to have a cytoplasmic localization in older animals. In adult testes, CK2α’ and CK2β subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2β is highly expressed in spermatogonia, whereas CK2α’ is barely detectable. Mature epididymal spermatozoa express CK2α’ in the acrosome and CK2β in the flagellum. This new evidence therefore indicates that protein kinase CK2 has a possible role at various stages during mammalian spermatogenesis, a process that involves proliferation, meiosis, apoptosis, and differentiation. CK2 might thus emerge as a new pivotal control enzyme at various levels in mammalian spermatogenesis.     

αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide

Amyloid fibril formation is associated with diseases such as Alzheimer’s, Parkinson’s, and prion diseases. Inhibition of amyloid fibril formation by molecular chaperone proteins, such as the small heat-shock protein αB-crystallin, may play a protective role in preventing the toxicity associated with this form of protein misfolding. Reduced and carboxymethylated κ-casein (RCMκ-CN), a protein derived from milk, readily and reproducibly forms fibrils at physiological temperature and pH. We investigated the toxicity of fibril formation by RCMκ-CN using neuronal model PC12 cells and determined whether the inhibition of fibril formation altered its cell toxicity. To resolve ambiguities in the literature, we also investigated whether fibril formation by amyloid-β1–40 (Aβ_1–40), the peptide associated with Alzheimer’s disease, was inhibited by αB-crystallin and if this affected the toxicity of Aβ. To this end, either RCMκ-CN or Aβ_1–40 was incubated at neutral pH to induce fibril formation before treating PC12 cells and assessing cell viability. Incubated (fibrillar) RCMκ-CN was more toxic to PC12 cells than native RCMκ-CN with the highest level of toxicity being associated with mature fibrils and protofibrils. Furthermore, the toxicity of RCMκ-CN was attenuated when its fibril formation was inhibited, either through the chaperone action of αB-crystallin or when it interacted with its natural binding partners in milk, α_S- and β-casein. Likewise, incubating Aβ_1–40 with αB-crystallin inhibited both Aβ_1–40 fibril formation and the associated cell toxicity. Importantly, by inhibiting fibril formation, αB-crystallin prevents the cell toxicity associated with protein misfolding.     

Comparative Characterization of Laminarinases from the Filamentous Marine Fungi Chaetomium Indicum Corda and Trichoderma Aureviride Rifai

Marine filamentous fungi (103 strains) isolated from various marine habitats were studied for their ability to produce extracellular O-glycosylhydrolases. Cultural filtrates of these strains were shown to contain a series of glycanases (laminarinases, amylases, cellulases, pustulanases) and glycosidases (β-glucosidases, N-acetyl-β-glucosaminidases, β-galactosidases, α-mannosidases).Two species of marine fungi from different habitats were chosen for isolation of laminarinases and detailed study on enzyme properties. The fungus Chaetomium indicum associated with the alga Fucus evanescens C. Agardh was collected near the Kuril Islands, and T. aureviride was sampled from bottom deposits of South China Sea. Properties of extracellular laminarinases were similar: temperature optimums (40–45 ^∘C), molecular masses (54–56 kDa), K _m (0.1–0.3 mg ml⁻¹). Temperature stability of laminarinase of C. indicum was significantly higher than those from T. aureveride. It is shown that these enzymes are specific to β-1,3-bonds in glucans, release predominantly glucose from laminaran and do not catalyze reaction of transglycosylation. Accoding to these data enzymes are exo-1,3-β-D-glucan-glucanohydrolases (EC 3.2.1.58). Inhibitor analysis demonstrated the significant role of tryptophan and tyrosine residues in the catalytic activity of enzymes. Molecules of T. aureviride laminarinase contained the functionally important thiol group.     

Studies on cell wall loosening enzymes in hormone treated internodes of <Emphasis Type="Italic">Merremia emarginata</Emphasis>

The cell wall loosening enzymes viz. glycosidases, polygalacturonase and xylanase were analyzed in cytoplasmic and wall bound fractions extracted from control and hormone (GA₃ NAA, PAA) treated internodes, as they are known to play a key role in cell wall metabolism. Among the glycosidases, wall bound β-glucosidase and α-galactosidase activities were significantly correlated with age of control internodes. Cytoplasmic α-galactosidase showed significant correlation in hormone treated internodes. Maximum correlation was observed in GA₃, followed by PAA and NAA. Wall bound xylanase activity was well correlated with length only in NAA treated internodes and less after GA₃ treatment while cytoplasmic xylanase showed correlation with intrnode length only in control and after NAA treatment. Cytoplasmic polygalacturonase showed correlation with internode length only after GA₃ treatment while wall bound polygalacturonase showed correlation with internode length after NAA treatment. The possible role of these enzymes in internode development is discussed.     

Effects of Yariv dyes, arabinogalactan-protein binding reagents, on the growth and viability of Brazilian pine suspension culture cells

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several aspects of plant growth and development. (β-d-glucosyl)₃ Yariv phenylglycoside (β-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures of Araucaria angustifolia, the Brazilian pine, with β-GlcY and observed inhibition of biomass increase in a culture medium with 50 μM β-GlcY. However, the growth was not inhibited by (α-d-galactosyl)₃ Yariv phenylglycoside (α-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that β-GlcY severely affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic cell death, were not observed in β-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic shrinkage and condensation were observed in β-GlcY-treated cells. In addition, callose accumulation, which is another marker of PCD, was also observed in β-GlcY-treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture medium did not reverse the growth inhibition caused by β-GlcY. These data indicate that the β-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven by necrosis.     

Selection of a standard culture medium for primary culture of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> Amebocytes

Summary This study provides information relevant to future research aimed at producing Limulus Amebocyte Lysate (LAL) in vitro, which would potentially reduce the need to harvest and bleed horseshoe crabs as in the current methods of LAL production. To address the need for primary culture of horseshoe crab amebocytes, this study tested the effects of a variety of standard insect cell culture media on amebocyte morphology and viability after 7 d of maintenance. Amerbocyte morphology was least altered from in vivo form in Grace’s Modified Insect Medium, with no observed degranulation of cells, as compared to the other media tested. There were significant differences in amebocyte viability among the six insect cell culture media tested. Grace’s Modified Insect Medium sustained viability of 77.2±5.1% (mean ± standard deviation) of amebocytes, followed distantly by Grace’s Insect Medium with 35.1±8.7% amebocyte viability. Results indicate that Grace’s Modified Insect Medium with horseshoe crab serum supplementation was the best candidate of the six media tested for future medium optimization for Limulus amebocyte requirements.     

Biochemical Changes Associated with Brassica juncea Seed Development, II: Glycosidases

Changes in α- and β-galactosidase, glucosidase, and α-mannosidase and β-xylosidase activities were analyzed in developing mustard (Brassica juncea) seed. A cubic polynomial described the seed dry weight data adequately. A close parallelism between the water content and dry matter accumulation was shown (r= 0.991). Glycosidase activities were detected in both cytoplasmic and wall fractions. The trend was similar in both of the fractions, but the activity of α-mannosidase and α-galactosidase was considerably greater in the wall-bound fraction. The water content and the activity of glycosidic enzymes showed a significant linear correlation (p < 0.001). The results suggest that glycosidases have an important role in cell wall loosening during mustard seed development. Received July 10, 1997; accepted January 28, 1998