Enzyme Replacement Therapy for Mucopolysaccharidosis IIID using Recombinant Human α-N-Acetylglucosamine-6-Sulfatase in Neonatal Mice.

There is currently no cure or effective treatment available for mucopolysaccharidosis type IIID (MPS IIID, Sanfilippo syndrome type D), a lysosomal storage disorder (LSD) caused by the deficiency of α-N-acetylglucosamine-6-sulfatase (GNS). The clinical symptoms of MPS IIID, like other subtypes of Sanfilippo syndrome, are largely localized to the central nervous system (CNS), and any treatments aiming to ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease need to be delivered across the blood-brain barrier. Here, we report a proof-of-concept enzyme replacement therapy (ERT) for MPS IIID using recombinant human α-N-acetylglucosamine-6-sulfatase (rhGNS) via intracerebroventricular (ICV) delivery in a neonatal MPS IIID mouse model. We overexpressed and purified rhGNS from CHO cells with a specific activity of 3.9 × 104 units/mg protein and a maximal enzymatic activity at lysosomal pH (pH 5.6), which was stable for over one month at 4 °C in artificial cerebrospinal fluid (CSF). We demonstrated that rhGNS was taken up by MPS IIID patient fibroblasts via the mannose 6-phosphate (M6P) receptor and reduced intracellular glycosaminoglycans to normal levels. The delivery of 5 µg of rhGNS into the lateral cerebral ventricle of neonatal MPS IIID mice resulted in normalization of the enzymatic activity in brain tissues; rhGNS was found to be enriched in lysosomes in MPS IIID-treated mice relative to the control. Furthermore, a single dose of rhGNS was able to reduce the accumulated heparan sulfate and ß-hexosaminidase. Our results demonstrate that rhGNS delivered into CSF is a potential therapeutic option for MPS IIID that is worthy of further development.

Heparan sulfate subdomains that are degraded by Sulf accumulate in cerebral amyloid ß plaques of Alzheimer's disease: evidence from mouse models and patients.

Alzheimer's disease (AD) is characterized by extracellular cerebral accumulation of amyloid ß peptide (Aß). Heparan sulfate (HS) is a glycosaminoglycan that is abundant in the extracellular space. The state of sulfation within the HS chain influences its ability to interact with a variety of proteins. Highly sulfated domains within HS are crucial for Aß aggregation in vitro. Here, we investigated the expression of the sulfated domains and HS disaccharide composition in the brains of Tg2576, J20, and T41 transgenic AD mouse models, and patients with AD. RB4CD12, a phage display antibody, recognizes highly sulfated domains of HS. The RB4CD12 epitope is abundant in the basement membrane of brain vessels under physiological conditions. In the cortex and hippocampus of the mice and patients with AD, RB4CD12 strongly stained both diffuse and neuritic amyloid plaques. Interestingly, RB4CD12 also stained the intracellular granules of certain hippocampal neurons in AD brains. Disaccharide compositions in vessel-enriched and nonvasculature fractions of Tg2576 mice and AD patients were found to be comparable to those of non-transgenic and non-demented controls, respectively. The RB4CD12 epitope in amyloid plaques was substantially degraded ex vivo by Sulf-1 and Sulf-2, extracellular HS endosulfatases. These results indicate that formation of highly sulfated HS domains may be upregulated in conjunction with AD pathogenesis, and that these domains can be enzymatically remodeled in AD brains.

Liquid chromatography-tandem mass spectrometry assay for the quantitation of plagiochin E and its main metabolite plagiochin E glucuronides in rat plasma.

Plagiochin E, a macrocyclic bisbibenzyl isolated from liverwort Marchantia polymorpha, was found to have antifungal activity. To evaluate the pharmacokinetics of plagiochin E in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantitation of plagiochin E and its total conjugated metabolites in rat plasma. For detection, a Sciex API 4000 LC-MS/MS with a TurboIonSpray ionization (ESI) inlet in the negative ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pretreated by a simple liquid-liquid extraction with ethyl acetate. The concentration of plagiochin E parent form was determined directly and the concentration of plagiochin E conjugated metabolites was assayed in the form of plagiochin E after treatment with beta-glucuronidase/sulfatase. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.5-1000.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.5 ng/mL for plagiochin E in 50 microL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of plagiochin E in rats after an oral and an intravenous administration.

Interest of new alkylsulfonylhydrazide-type compound in the treatment of alcohol use disorders.

RATIONALE: Recent preclinical research suggested that histone deacetylase inhibitors (HDACIs) and specifically class I HDAC selective inhibitors might be useful to treat alcohol use disorders (AUDs). OBJECTIVE: The objective of this study was to find a new inhibitor of the HDAC-1 isoenzyme and to test its efficacy in an animal model of AUDs. METHODS: In the present study, we prepared new derivatives bearing sulfonylhydrazide-type zinc-binding group (ZBG) and evaluated these compounds in vitro on HDAC-1 isoenzyme. The most promising compound was tested on ethanol operant self-administration and relapse in rats. RESULTS: We showed that the alkylsulfonylhydrazide-type compound (ASH) reduced by more than 55% the total amount of ethanol consumed after one intracerebroventricular microinjection, while no effect was observed on motivation of the animals to consume ethanol. In addition, one ASH injection in the central amygdala reduced relapse. CONCLUSIONS: Our study demonstrated that a new compound designed to target HDAC-1 is effective in reducing ethanol intake and relapse in rats and further confirm the interest of pursuing research to study the exact mechanism by which such inhibitor may be useful to treat AUDs.

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi.

Novel antifoulants: inhibition of larval attachment by proteases.

We investigated the effect of commercially available enzymes (alpha-amylase, alpha-galactosidase, papain, trypsin, and lipase) as well as proteases from deep-sea bacteria on the larval attachment of the bryozoan Bugula neritina L. The 50% effective concentrations (EC(50)) of the commercial proteases were 10 times lower than those of other enzymes. Crude proteases from six deep-sea Pseudoalteromonas species significantly decreased larval attachment at concentrations of 0.03 to 1 mIU ml(-1). The EC(50) of the pure protease from the bacterium Pseudoalteromonas issachenkonii UST041101-043 was close to 1 ng ml(-1) (0.1 mIU ml(-1)). The protease and trypsin individually incorporated in a water-soluble paint matrix inhibited biofouling in a field experiment. There are certain correlations between production of proteases by bacterial films and inhibition of larval attachment. None of the bacteria with biofilms that induced attachment of B. neritina produced proteolytic enzymes, whereas most of the bacteria that formed inhibitive biofilms produced proteases. Our investigation demonstrated the potential use of proteolytic enzymes for antifouling defense.

Metabolism of pyrene by aquatic crustacean, Daphnia magna.

The aquatic crustacean Daphnia magna is an important species for ecotoxicological study, and is often used as a test organism for environmental risk assessment. However, the mechanism of xenobiotic metabolism by this species has not been studied in detail. In the present study, pyrene was used as model substance to investigate the mechanism of xenobiotic metabolism in D. magna. The results of 24-h exposure experiments showed that D. magna could metabolize pyrene and biotransform it into water-soluble metabolites. On the other hand, the metabolism of pyrene was significantly inhibited by SKF-525A as the cytochrome P450 (CYP) inhibitor. These observations indicated that oxidation by CYP participated in the biotransformation of pyrene by D. magna. We also identified the pyrene metabolites formed by D. magna by HPLC with an electrospray ionization triple quadrupole mass spectrometry detector (LC/ESIMS/MS) and de-conjugation by sulfatase, beta-glucuronidase, and beta-glucosidase. One of the metabolites was ionized in ESI negative mode and formed a dominant mass of m/z 297 (MS) with the product ion of m/z 217 (MS2). Furthermore, this metabolite formed 1-hydroxypyrene on treatment with sulfatase. This metabolite was considered to be a sulfate conjugate of oxidized pyrene (1-hydroxypyrenesulfate). Furthermore, we quantified the deconjugated 1-hydroxypyrene formed by the above enzyme treatment. It showed that 52% of the total metabolized pyrene was biotransformed into 1-hydroxypyrene-sulfate, and more than 73% was biotransformed into oxidized pyrene conjugate. These results indicated that CYP and several conjugation enzymes participate in its biotransformation, and sulfation is important in D. magna for metabolism and elimination of xenobiotics.

Difference in plasma metabolite concentration after ingestion of lemon flavonoids and their aglycones in humans.

The concentrations of metabolites in human plasma after ingestion of flavanone glycosides (FG) and their aglycones (FA) in lemon were examined. FG consisting abundantly of eriocitrin were prepared from lemon peel and FA consisting abundantly of eriodictyol were prepared from FG by treatment with beta-glucosidase. Eriodictyol, homoeriodictyol, and hesperetin in plasma up to 4 h after ingestion of FG with water or FA with water by subjects were not detected in plasma of non-enzyme treatment but in plasma after treatment with beta-glucronidase and sulfatase. Metabolites in plasma after ingestion of FG and FA in humans were shown to exist as the glucuro- and/or sulfo-conjugates of eriodictyol, homoeriodictyol, and hesperetin. After ingestion of FA, the concentration of metabolites in plasma exhibited a high maximum peak at 1 h. The AUC (area under the blood concentration time curve) level of metabolites of FA was higher than that of FG. FA were suggested to be absorbed faster and in higher amounts than FG. The AUC of metabolites in subject plasma after ingestion of FG with flavonoid-depleted lemon juice was shown to change to a low level in comparison with that of FG with water. The maximum concentration peak of metabolites in plasma was faster at 0.5 h than FA with water but the AUC level was similar to FA with water, when subjects ingested FA with vodka (40%, ethanol). The absorption hour of FG and FA was shown to be affected by the co-existing solution.

Chromatographic analyses of 3-methylcholanthrene metabolism in adult and fetal mice and the occurrence of conjugating enzymes in the fetus.

Adult pregnant mice were given i.v. injections of (3H)3-methylcholanthrene (20 muCi in 1.1 mug/mouse) or (14C)3-methylcholanthrene (1.0 muCi in 48 mug/mouse). Ethanol extracts of their tissues were chromatographed on Sephadex LH-20. Three groups of 3-methylcholanthrene metabolites were obtained: one group as yet unidentified, one containing the hydrocarbon and hydroxylated derivatives, and a third consisting of conjugated metabolites from the treated adult mice and their fetuses. The conjugated metabolites in tissue and in bile were separated into two fractions; one was acted on by beta-glucuronidase and to a lesser extent by arylsulfatase, and the other was resistant to these enzymes but completely susceptible to acid hydrolysis. The hydrolysis resulted in altered chromatographic behavior characteristic of the hydroxy compounds, which also appear in tissue. The enzyme-resistant conjugates were predominant in brain, muscle, and lung, and the enzyme-labile conjugates were predominant in the kidney, liver, and bile of adult mice. These conjugated metabolites were also demonstrated in fetal mice; some appeared in the fetus as early as the thirteenth day of gestation, the most immature fetus so far examined. The resistant group was predominant in the early developmental stages of the fetus and the susceptible group was increased in the excretory organs such as the kidney, liver, and contents of the intestinal tract as the fetuses approached term. transplacental transfer of conjugated metabolites from the mother to the fetus did not take place, although the parent 3-methylcholanthrene and its nonconjugated metabolites were transferred. We therefore assume that drug-metabolizing enzymes, including hydroxylases and conjugases, are active in the fetal mouse tissues as well as in the adult.

Cyclophosphamide metabolism in children.

The alkylating agent cyclophosphamide is a prodrug which is metabolized in vivo to produce both therapeutic and toxic effects. Cyclophosphamide metabolism was investigated in 36 children with various malignancies. Concentrations of cyclophosphamide and its principal metabolites were measured in plasma and urine using a quantitative high-performance TLC method. The results indicated a high degree of inter-patient variation in metabolism. In contrast to previous adult studies on urinary metabolites, plasma carboxyphosphamide concentrations did not support the existence of polymorphic metabolism. Plasma concentrations of dechlorethylcyclophosphamide and carboxyphosphamide were correlated in individual patients, suggesting that the activity of both aldehyde dehydrogenase and cytochrome P450 enzyme(s) determine carboxyphosphamide production in vivo. The presence of ketocyclophosphamide in plasma was strongly associated with dexamethasone pretreatment and was also accompanied by a high clearance of the parent drug. Interpatient differences in metabolism reflect individual levels of enzyme expression and may contribute to variation in clinical effect.

A radioimmunoassay for measurement of thyronine and its acetic acid analog in urine.

A sensitive and specific RIA has been developed to measure thyronine (To) in urine. The RIA used an anti-To antibody obtained from a rabbit immunized with a L-To-human serum albumin conjugate and [3H]To as the radioligand. The acetic acid analog of To (ToAc), that is the diphenyl structure with an acetic acid side-chain, cross-reacted strongly with the antibody. Relative to To, it cross-reacted 160% in phosphate-buffered saline, pH 7.4, and 100% in 0.075 mol/L barbital buffer, pH 8.6, containing sodium salicylate (final concentration, 8 mg/mL). The latter conditions were employed for the RIA, and the results reported thus reflect the presence of To and/or ToAc. 3-Monoiodothyronine, 3'-monoiodothyronine, 3',5'-diiodothyronine, and 3,5-diiodothyronine cross-reacted with the anti-To antibody 1.9%, 1.7%, 0.3%, and 0.2%, respectively; the cross-reactivity of other To derivatives and tyrosine and its derivatives was less than 0.05%. Urinary To and/or ToAc excretion in 12 normal subjects averaged 16 +/- 2 (+/- SE) micrograms/day (59 +/- 9 nmol/day) or 14 +/- 2 micrograms/g creatinine (5.9 +/- 0.6 nmol/mmol creatinine). Treatment of urine from normal subjects with beta-glucuronidase or sulfatase did not significantly alter the To content. Column and thin layer chromatographic studies revealed that 83% and 61%, respectively (range, 37-100%), of urinary To immunoreactivity was attributable to ToAc. The mean daily excretion of To in 20 patients with nonthyroidal illness [NTI; 22 +/- 4 micrograms/day (82 +/- 17 nmol/day)] was similar to that in normal subjects, but was elevated when expressed as nanomoles per mmol creatinine (20 +/- 2; P less than 0.001), because creatinine excretion was reduced in the NTI patients. The mean daily urinary To excretion in 13 patients with hyperthyroidism due to Graves' disease was slightly elevated [29 +/- 6 micrograms/day (108 +/- 21 nmol/day); P less than 0.1], but was clearly elevated when expressed as nanomoles per mmol creatinine (37 +/- 8; P less than 0.001), again because creatinine excretion was reduced in these patients. The mean urinary To excretion was subnormal in 13 patients with hypothyroidism and was significantly (P less than 0.005) less than that in the NTI patients regardless of the manner in which the results were expressed. Analysis of pronase hydrolysates of thyroid glands obtained at autopsy from euthyroid patients suggested that the To content of the thyroid approximates only 1.2% that of T4, supporting the thesis that prior iodination of tyrosine is critical for the coupling process in the thyroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticosterone storage within the adrenal cortex: evidence for a sulphate conjugate.

Incubation of homogenized rat adrenal glands with sulphatase for 18 h resulted in a significant increase in the amount of unconjugated corticosterone that could be extracted from the gland and which could not be attributed to de-novo synthesis of corticosterone during the incubation. Therefore corticosterone may be stored within the adrenal gland as a sulphate conjugate rather than as unconjugated hormone. Exposure of animals to footshock stress resulted in an increase in the amount of unconjugated corticosterone in the gland and a disappearance of the conjugate form. The rapid disappearance of the sulphate conjugate may reflect the activation of a steroid sulphatase by ACTH. However, the release of corticosterone from a storage form such as corticosterone sulphate could not explain entirely the initial increase in plasma corticosterone concentration following the stress. It was calculated that a pair of adrenal glands would have to store up to 20 nmole corticosterone in order to account for the initial increase in plasma corticosterone. This level was much greater than that extracted from a pair of glands (4.8 nmol) even after sulphatase pretreatment.

Differential modification of opiate receptor activity by arylsulfatase treatment.

Treatment with the enzyme arylsulfatase in vivo selectively attenuated the effect of analgesia induced by morphine, beta-endorphin or ethylketocyclazocine but not that induced by Sandoz FK33824 or D-ala2-D-leu5-enkephalin. The effect on morphine analgesia was indicated both by an increased morphine ED50 in the presence of a fixed dose of naloxone and by a decreased naloxone ED50 in the presence of a fixed dose of morphine. Arylsulfatase treatment in vivo also selectively affected in vitro ligand binding; Bmax values of the low affinity binding site of dihydromorphine, naloxone, D-ala2-D-leu5-enkephalin, D-ala2-met5-enkephalinamide and ethylketocyclazocine were decreased significantly while the Bmax values of the high affinity sites as well as the KD values of both the high and low affinity sites were affected little or not at all. The data suggest that the change induced by the enzyme may have been due to the alteration of certain constituents of the low affinity opiate binding site.

Caprine mucopolysaccharidosis IIID: a preliminary trial of enzyme replacement therapy.

Mucopolysaccharidosis type IIID (MPS IIID) is a lysosomal storage disorder resulting from lack of activity of the lysosomal hydrolase N-acetylglucosamine 6-sulfatase (6S) (EC 3.1.6.14). The syndrome is associated with systemic and central nervous system (CNS) heparan sulfate glycosaminoglycan (HS-GAG) accumulation, secondary storage of lipids, and severe, progressive dementia. In this investigation, caprine MPS IIID, established as a large animal model for the human disease, was used to evaluate the efficacy of enzyme replacement therapy (ERT). Recombinant caprine 6S (rc6S) (1 mg/kg/dose) was administered intravenously to one MPS IIID goat kid at 2, 3, and 4 wks of age. Five days after the last dose, the uronic acid (UA) content and the composition of uncatabolized HS-GAG fractions in the brain of the ERT-treated MPS IIID kid were similar to those from a control, untreated MPS IIID animal. However, hepatic uronic acid levels in the treated MPS IIID kid were approximately 90% lower than those in the untreated MPS IIID control; whereas the composition of the residual hepatic HS-GAG was identical to that in the untreated animal. Marked reduction of lysosomal storage vacuoles in hepatic cells of the treated MPS IIID kid was observed, but ERT had no effect on CNS lesions. No residual 6S activity was detected in brain or liver. This preliminary investigation indicates that other treatment regimens will be necessary to ameliorate MPS III-related CNS lesions.

Glycation of human tissue and serum creatine kinase.

Human creatine kinase (CK) was demonstrated to be partly present as a glycated molecule. Sialic acid, galactose and sulfate were also found to be present on the molecule. The glycated forms were characterized by higher activation energies and were thermally unstable. Skeletal muscle CK showed lower relative binding towards the lectin concanavalin A (Con A) in comparison with the heart tissue forms. After skeletal muscle trauma, CK in serum was found to be less glycated than in tissue. After acute myocardial infarction (AMI), no glycated CK could be detected in serum. Following injury, it appears that the transfer from tissue to plasma is accompanied by a loss of glycated isoforms. High-voltage electrophoresis showed no differences in the distribution of CK isoforms between the glycated and non-glycated forms.

Soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone Haliotis tuberculata: extraction and partial analysis of nacre proteins.

Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and beta-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.

Synthesis and biochemical evaluation of some novel benzoic acid based esters as potential inhibitors of oestrone sulphatase.

Oestrone sulphatase is an important target in the fight against hormone-dependent breast cancer. In an effort to investigate the reported definitive pharmacophore for oestrone sulphatase and continue our search for potent inhibitors of this enzyme, we have undertaken extensive synthesis, biochemical evaluation and physicochemical property determination of a range of benzoic acid based esters. Here, we report the initial results of our study into a series of straight chain alkyl esters of 4-sulphonylbenzoic acid. Using these compounds, we have investigated the involvement of two physicochemical properties, namely logP and pK(a). The results of this study show that there was a strong correlation between the inhibitory activity and the logP of the parent compound. Within the series of compounds studied, hydrophobicity appears to be a more important factor than pK(a) in determining the overall inhibitory activity. In a previous report, we showed that pK(a) plays an important role in stabilizing the phenoxide ion resulting from the hydrolysis of the sulphamate group. Here, we propose that although pK(a) is an important factor in determining the overall inhibitory activity when a wide range of compounds are considered, both hydrophobicity and pK(a) need to be considered in the design of potential inhibitors of oestrone sulphatase.

[Effect of enzymes on the adhesion of fungi of the genus Candida to the buccal mucosa in vitro]. / Wplyw enzymów na przyleganie grzybów z rodzaju Candida do komórek blony sluzowej policzka in vitro.

Influence of selected enzymes as pepsin, pronase, lysozyme, and glusulase on adhesion of 15 strains of Candida sp. to buccal epithelial cells of oral cavity of man was examined in vitro. The enzymes were used in such concentration which did not influence the viability of fungal cells. Only pepsin preincubation had no influence on adhesion test, the remaining enzymes inhibited significantly attachment of Candida strains to epithelial cells in an adherence assay in vitro.