Effects of cortisone and thyroxine on suckling rat liver N-acetyl-ß-glucosaminidase isozymes: Comparison with adult reactivity

Suckling rat liver N-acetyl-β-glucosaminidase (hexosaminidase) activity undergoes considerable fluctuation during the first two weeks of life. As two major forms of hexosaminidase (A, heat-labile, and B, heat-stable) are known to exist in both human and adult rat liver, we choose to examine the effect of the maturative hormones, thyroxine and cortisone, upon these isozymes during the suckling period. Between days 7 and 15, the observed developmental change is attributable solely to an increase in the ‘A-like’ (heat-labile) form of the enzyme; an enhanced response is seen in thyroxine-injected 11–15-day old animals. The response may be considered ‘age-independent’, as adult animals react in the same manner. In contrast, cortisone-injected sucklings show a decrease in both A and B isozymes, while in adults no changes in total activity or isozyme distribution are evoked. The ratio of hexosaminidase A to hexosaminidase B in suckling rat liver appears to shift in favor of the labile (A) isozyme early in development.     

PURIFICATION AND PROPERTIES OF BRAIN N-ACETYL-β-HEXOSAMINIDASE A

—N-Acetyl-β-hexosaminidase A was purified to homogeneity from human and monkey brains by the conventional procedures followed by concanavalin A–Sepharose affinity chromatography. The optimal activity was observed at pH 4·5 for both enzyme preparations with both the aglycones N-acetylglucosamine and N-acetylgalactosamine derivatives. The K_m values for hexosaminidase A from monkey brain were 0·26 mm and 0·04 mm respectively for N-acetylglucosamine and N-acetylgalactosamine. K_m values obtained for glucosamine and galactosamine derivatives for the human brain hexosaminidase A were of the same order. The glycoprotein nature of the enzymes was established by the affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the enzyme molecule from monkey brain.     

Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.     

Assignment of the human gene for hexosaminidase B to chromosome 5

Mouse-human somatic cell hybrids between different mouse and human cells were studied for the expression of human hexosaminidases A and B activities. The expression of human hexosaminidase B in the hybrids was found to segregate concordantly with the presence of the human chromosome 5. Mouse-human hybrid clones containing either the human chromosomes 5 and 7 only or the human chromosome 7 only were also included in this study. Expression of human hexosaminidase B activity was detected only in those clones containing human chromosome 5. These results indicate that the gene(s) for human hexosaminidase B is located on chromosome 5. No hexosaminidase A activity was detected in clones which contained either human chromosomes 5 and 7 or chromosome 7.     

Genetic variation of hexosaminidase A and arylsulfatase A activity. Correlation study in amnio-maternal paris of cultured cells

Summary Pairs of cultured amniotic cells and maternal fibroblasts (feto-maternal pairs) were studied for hexosaminidase A (HXA) and arylsulfatase A (ASA) activity. These lysosomal enzyme activities are genetically deficient in Tay-Sachs disease and metachromatic leukodystrophy, respectively. After HXA was standardized by relating it to hexosaminidase B (HXB) activity, a feto-maternal correlation coefficient of r=0.51 (n=32; 95% confidence limits 0.197–0.73) was found for the HXA/HXB activity quotients. This coefficient was near the 0.5 value theoretically valid for mother-child pairs, suggesting that the studied activities reflect essentially the genetic variability. The studies of ASA revealed a high variability of individual activities, which was reduced in two steps: (1) The ASA activity was related to the mean of two lysosomal reference enzyme activities, total hexosaminidase and acid -galactosidase. (2) Since the square root of ASA activity was found to follow more closely the variation of the reference activities, the square root of ASA activity over the mean reference activity was taken as a more standardized measure of ASA activity, and the quotient was treated statistically. Positive feto-maternal correlation of standardized ASA activity was obtained after the elimination of three pairs with extreme values. A correlation coefficient of r=0.42 (n=26; 95% confidence limits 0.039–0.695) resulted.The implications of these correlation studies for the problem of heterozygote identification by quantitative enzyme assays in families deficient in HXA and ASA activity were considered.     

Antibody against purified human hexosaminidase B cross-reacting with human hexosaminidase A

Human hexosaminidase B has been purified to virtually homogeneous state from placenta. An anti-serum has been prepared in rabbits against the purified preparation. The serum reacted equally with human hexosaminidase B (free of hexosaminidase A) and with human hexosaminidase A (free of hexosaminidase B) as shown by immunodiffusion and by precipitation of enzyme activity from solution.     

Purification and properties of the hexosaminidase A-activating protein from human liver

Human liver extracts contain an activating protein which is required for hexosaminidase A-catalysed hydrolysis of the N-acetylgalactosaminyl linkage of G_M2 ganglioside [N-acetylgalactosaminyl-(N-acetylneuraminyl) galactosylglucosylceramide]. A partially purified preparation of human liver hexosaminidase A that is substantially free of G_M2 ganglioside hydrolase activity is used to assay the activating protein. The proceudres of heat and alcohol denaturation, ion-exchange chromatography and gel filtration were used to purify the activating protein over 100-fold from crude human liver extracts. When the purified activating protein is analysed by polyacrylamide-gel disc electrophoresis, two closely migrating protein bands are seen. When purified activating protein is used to reconstitute the G_M2 ganglioside hydrolase activity, the rate of reaction is proportional to the amount of hexosaminidase A used. The activation is specific for G_M2 ganglioside and and hexosaminidase A. The activating protein did not stimulate hydrolysis of asialo-G_M2 ganglioside by either hexosaminidase A or B. Hexosaminidase B did not catalyse hydrolysis of G_M2 ganglioside with or without the activator. Kinetic experiments suggest the presence of an enzyme–activator complex. The dissociation constant of this complex is decreased when higher concentrations of substrate are used, suggesting the formation of a ternary complex between enzyme, activator and substrate. Determination of the molecular weight of the activating protein by gel-filtration and sedimentation-velocity methods gave values of 36000 and 39000 respectively.     

Concanavalin A-Sepharose Chromatography of Hexosaminidase Isozymes Secreted By Tetrahymena*

Tetrahymena pyriformis strain HSM secretes 4 isozymes of hexosaminidase. Purified isozymes B₁ and B₂ are eluted from the void volume of a concanavalin A-Sepharose column, suggesting that they are not glycosylated. Purified isozymes A₁ and A₂ bind to the column and are eluted at ～0.1 M α-methylmannoside, suggesting that these isozymes are glycoproteins. In agreement with earlier deductions based on a differential kinetic assay for the A and B isozymes, the elution pattern of hexosaminidase activity from material secreted by cells grown to early and late stationary phase was consistent with these secretions containing primarily the B and the A isozymes, respectively.     

Immunological properties of N-acetyl-β-d-glucosaminidase of normal human liver and of GM2-gangliosidosis liver

Antisera were raised to a partially purified preparation of human liver hexosaminidase and to highly purified preparations of hexosaminidase isoenzymes A and B. All the antisera precipitated the enzyme in an enzymically active form, which could be located on immunodiffusion and immunoelectrophoretic gels by using a histochemical substrate. The antisera to the purified isoenzymes were shown to react with hexosaminidase from human liver, kidney, brain and spleen, but did not cross-react with human liver beta-glucosidase, beta-galactosidase, alpha-mannosidase, beta-xylosidase, arylsulphatase or acid phosphatase. Hexosaminidases A and B were immunologically identical. The immunological properties of the hexosaminidases from livers of patients with three types of GM(2)-gangliosidoses were closely similar. No evidence could be found for cross-reacting material in enzyme-deficient states.     

Diagnosis of infantile and juvenile forms of GM2 gangliosidosis variant 0. residual activities toward natural and different synthetic substrates

Summary p-Nitrophenyl-6-sulfo-2-acetamido-2-deoxy--d-glucopyranoside, which is known to be a specific substrate for human hexosaminidase A, has recently been used successfully for diagnosis of variants B and B¹ of G_M2-gangliosidosis (Fuchs et al. 1983; Kytzia et al. 1983; Li et al. 1983). However, it is hydrolyzed by hexosaminidase S as well and is therefore not suitable for detection of patients with variant 0, who reach the normal range of activity toward this substrats. Assay of ganglioside G_M2 cleaving activity in fibroblast extracts in the presence of the natural G_M2 activator protein reveals residual hexosaminidase A activities of less than 2% of normal controls in two infantile and up to 7.5% in two juvenile patients with variant 0.     

Release of Mediator Enzyme β-Hexosaminidase and Modulated Gene Expression Accompany Hemocyte Degranulation in Response to Parasitism in the Silkworm Bombyx mori

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme β-hexosaminidase showed increased activity and expression of β-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R²?=?0.71) between LDH activity and β-hexosaminidase released after the infection showed parasitic–protein-induced hemocyte damage and accompanied release of the enzymes. Expression of β-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R²?=?0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45–60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

     

Introduction of the alpha subunit mutation associated with the B1 variant of Tay-Sachs disease into the beta subunit produces a beta-hexosaminidase B without catalytic activity

Lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (alpha beta) and hexosaminidase B (beta beta). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the alpha subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g. GM2 ganglioside. Recently, a point mutation, producing a single amino acid substitution in the alpha subunit (Arg178-His), has been found to be associated with the B1 variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward alpha subunit-specific substrates. However, because of the presence of an active beta subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected alpha subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the alpha and beta subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human beta subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on beta subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that beta homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe B1 phenotype results from a totally inactive alpha-subunit in hexosaminidase A.     

5-HYDROXYTRYPTAMINE CATABOLISM IN THE RAT BRAIN DURING ONTOGENESIS

Although the serotoninergic innervation is immature in the brains of young rats, the 5-HIAA content is similar to that found in adults. As indicated by the ratio of 5-HIAA to 5-HT levels in the brain stem and the forebrain, the catabolism of the indolamine was more rapid during the first 3 postnatal weeks than in adults. This was contirmed by measuring the total formation of [³H]5-HIAA from [³H]5-HT newly synthesized from L-[³H]tryptophan in brain stem slices of young and adult rats. Electrolytic lesions of midbrain raphe nuclei (B7 and B8) performed on the 5th postnatal day resulted in parallel decreases in brain 5-HT and 5-HIAA levels; this ruled out the possibility that 5-HIAA might be formed from 5-HT synthesized outside serotoninergic neurons, using peripheral 5-hydroxytryptophan. Inhibition of 5-HT storage by reserpine pretreatment did not alter the higher capacity of newborn tissues to catabolize exogenous [³H]5-HT. Therefore, possible differences in 5-HT binding in serotoninergic neurons between newborn and adult rats were not likely to account for the differences in 5-HT catabolism. Estimation of the rate of 5-HIAA efflux from the brain after MAO inhibition did not reveal marked changes with age. The activity of MAO type A, the enzyme involved in 5-HT catabolism, was higher during early life than later on. This could be shown by using 5-HT as substrate and clorgyline as a selective inhibitor. An opposite pattern of development was seen for MAO B, measured with benzylamine as substrate and deprenyl as selective inhibitor. These results suggest that the high 5-HIAA levels found in the brains of young rats can be attributed mainly to the presence of high MAO A activity during early life.     

OLIGOSACCHARIDE STORAGE IN BRAINS FROM PATIENTS WITH FUCOSIDOSIS,GM1-GANGLIOSIDOSIS AND GM2-GANGLIOSIDOSIS (SANDHOFF'S DISEASE)1

Abstract— Analysis of whole autopsy brain from a patient with fucosidosis (α-fucosidase deficiency) revealed minor storage of H-antigen glycolipid [Fuc (α, 1→2) Gal-GlcNAc-Gal-Glc-Ceramide] and a slightly abnormal ganglioside composition in the form of a two-fold elevation of G_M1 and the presence of a fucose-containing glycolipid (a minor component) which co-migrated with G_D1a. The major storage materials in fucosidosis brain were an oligosaccharide (Fuc-Gal-GlcNAc-Man[Fuc-Gal-GlcNAc-Man]-ManGlcNAc) and a disaccharide [Fuc(α, 1→6)-GlcNAc] in the approximate ratio of 5:1. Lesser amounts of a related oligosaccharide (Gal-GlcNAc-Man[Gal-GlcNAc-Man]-Man-GlcNAc) were isolated from the brain of patients with G_M1-gangliosidosis (Types I and II) where the major storage material is known to be G_M1-ganglioside (Gal (β, 1→3)GalNAc(β, 1→4) [NeuNAcf(α, 2→3) Gal(β, 1→4)Glc-Ceramide). Similarly, a related oligosaccharide (GlcNAc-Man [GlcNAc-Man]-Man-GlcNAc) was isolated from the brain of a patient with a total deficiency of N-acetyl-β-d -hexosaminidase (Sandhoff variant of G_M2-gangliosidosis) where the major storage products are known to be G_M2-ganglioside (GalNAc (β 1→4) [NeuNAc (α, 2→3)Gal(β, 1→4)Glc-Ceramine) and its asialo derivative. These studies indicate that glycoproteins containing at least 2 mol of l -fucose per oligosaccharide unit are normally catabolized in human brain. Further, it appears that such glycoproteins are initially catabolized by an endo-N-acetylglucosaminidase to release an oligosaccharide which is then degraded by the sequential action of exo-glycosidases.     

On the molecular basis of Sandhoff's disease

Summary The results of Hooghwinkel et al. (1972) concerning the existence of a third human N-acetylhexosaminidase, designated C, are confirmed. This hexosaminidase exhibits low activity and has therefore generally been overlooked until now. We suggest that the hexosaminidase A represents a heteromer consisting of B- and C-subunits. According to this assumption Sandhoff's disease would reflect a defect at the hexosaminidase B-locus, whereas Tay-Sachs' disease would be attributable to deficient C-subunits.

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Zusammenfassung Hooghwinkel et al. (1972) beschrieben kürzlich eine dritte N-acetyl-Hexosaminidase im menschlichen Gehirn. Diese Hexosaminidase C haben wir elektrophoretisch in Fibroblasten, Leukocyten und Gehirngewebe nachgewiesen. In Fibroblasten eines Patienten mit Sandhoffscher Krankheit erschien ihre Aktivit?t deutlich vermehrt. Wir sind der Ansicht, da? es sich bei der Hexosaminidase A um ein Heteromer aus B- und C-Untereinheiten handeln k?nnte; demnach w?re die Sandhoffsche Krankheit durch einen Defekt am Hexosaminidase B-Locus bedingt, w?hrend bei Tay-Sachs-Kranken C-Untereinheiten fehlten.

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Supported by the Deutsche Forschungsgemeinschaft.     

Presence of an atypical thermolabile species of beta-hexosaminidase B in metastatic-tumour tissue of human liver.

An atypical thermolabile species of Hex B (hexosaminidase B) has been found in metastatic-tumour sites of human liver which has a thermostability curve similar to that of Hex A (hexosaminidase A), which is present in decreased amounts relative to the Hex A isoenzyme, and which exhibits decreased relative activity at acidic pH values (2.6-3.6) when compared with control-liver Hex B. This atypical Hex B isoenzyme has a normal apparent Michaelis constant (0.6 mM) for 4-methylumbelliferyl 2-deoxy-2-acetamido-beta-D-glucopyranoside. The presence of this atypical Hex B suggests that variant beta-chains are being produced in metastatic-tumour tissue.     

Antibacterial and anti‐inflammatory activity of a temporin B peptide analogue on an in vitro model of cystic fibrosis

Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure‐activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram‐positive and Gram‐negative bacteria. In this paper, we investigated the antimicrobial and anti‐inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro‐inflammatory chemokines and cytokines interleukin (IL)‐8, IL‐1β, IL‐6 and tumor necrosis factor‐α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro‐inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of Non-Volatile Constituents of Elsholtzia ciliata (Thunb.) Hyl. from Southern Vietnam on Reactive Oxygen Species and Nitric Oxide Release in Macrophages

The extract of Elsholtzia ciliata aerial parts was subjected to bio-guided isolation using the intercellular ROS reduction in J774A.1 macrophages to monitor the anti-oxidative activity. Fifteen compounds were isolated from the active fractions including eleven flavonoids (vitexin, pedalin, luteolin-7-O-β-d -glucopyranoside, apigenin-5-O-β-d -glucopyranoside, apigenin-7-O-β-d -glucopyranoside, chrysoeriol-7-O-β-d -glucopyranoside, 7,3′-dimethoxyluteolin-6-O-β-d -glucopyranoside, luteolin, 5,6,4′-trihydroxy-7,3′-dimethoxyflavone, 5-hydroxy-6,7-dimethoxyflavone (compound 13 ), 5-hydroxy-7,8-dimethoxyflavone); three hydroxycinnamic acid derivatives (caffeic acid, 4-(E)-caffeoyl-l -threonic acid, 4-O-(E)-p-coumaroyl-l -threonic acid) and one fatty acid (α-linolenic acid). The biological evaluation of these compounds (10–2.5 μm ) indicated that all of them exerted good antioxidant and anti-inflammatory activities, in particular compound 13 .     

Expression of the Na+-d-Glucose Cotransporter SGLT1 in Neurons

Abstract: In brains of the rabbit, pig, and human, expression of the high-affinity Na⁺-d -glucose cotransporter SGLT1 and of the protein RS1, which alters the activity of SGLT1, was demonstrated. In situ hybridization showed that SGLT1 and RS1 are transcribed in pyramidal cells of brain cortex and hippocampus and in Purkinje cells of cerebellum. In neurons of pig brain SGLT1 protein was demonstrated by western blotting with synaptosomal membranes and by immunohistochemistry, which showed SGLT1 in pyramidal and Purkinje cells. To test whether SGLT1 in neurons may be activated during increased d -glucose consumption, an epileptic seizure was induced in rat brain, and the uptake of specific nonmetabolized substrates of SGLT1 {[¹⁴C]methyl-α-d -glucopyranoside ([¹⁴C]AMG)} and of Na⁺-independent transporters {2-deoxy-d -[¹⁴C]glucose([¹⁴C]2-DG)} was analyzed by autoradiography. During the seizure the uptake of AMG and 2-DG was increased in the focus. Within two hours after the seizure 2-DG uptake in the focus returned to normal. In contrast, the AMG uptake in the focus area was still increased 1 day later. The data show that the high-affinity Na⁺-d -glucose cotransporter SGLT1 is expressed in neurons and can be up-regulated.     

Three New 2,2′-Difurylketone Derivatives and Two New Chromones from the Rehmanniae Radix Praeparata

Rehmanniae Radix Praeparata is the processed products of the root of Rehmannia glutinosa. It has been used as a Traditional Chinese Medicine for thousands of years, and it has been found to possess widely pharmacological activities. In this study, three new 2,2′-difurylketone derivatives (rehmanniaeketone A–C) and two new chromones [3,8-dihydroxy-2-(2-hydroxyethyl)chromone and 3,8-dihydroxy-2-[(2-O-α-D-galactopyranosyloxy)ethyl]chromone] were isolated from the Rehmanniae Radix Praeparata. Furthermore all of the compounds were subjected to cytotoxic testing against the human lung carcinoma A549 cells. The cytotoxic results showed that rehmanniaeketone B and rehmanniaeketone C exhibited more stronger inhibition effects on the cell activity of A549 cells with the IC₅₀ 5.23 μM and 2.05 μM than other compounds. And 3,8-dihydroxy-2-(2-hydroxyethyl)chromone exhibited moderately inhibitory activity with the IC₅₀ 61 μM. Rehmanniaeketone A and 3,8-dihydroxy-2-[(2-O-α-D-galactopyranosyloxy]chromone showed no inhibitory effects.