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C Freeman

Publications and source records attributed to C Freeman.

At least 73 records · Page 4Linked to original sources
PubMed1992

Recalcitrant high-molecular-weight material, an inhibitor of microbial metabolism in river biofilms.

Recalcitrant high (>1,000)-molecular-weight materials (>1K materials) have been proposed to inhibit the metabolic activity of river biofilms. Evidence is presented to support the hypothesis that the mode of action is through occlusion of the surface of the biofilm by recalcitrant >1K materials. The evidence includes the following. (i) The phenomenon was particularly prominent in brown-water rivers, which are rich in recalcitrant >1K material. (ii)Temporal changes in inhibition intensity were observed, which coincided with seasonal changes in the relative recalcitrance of river water >1K materials. (iii) Stores of intracellular carbon (poly-beta-hydroxyalkanoates) were progressively compromised by increasing the supply of recalcitrant >1K materials. (iv) Nontoxic synthetic analogs of recalcitrant >1K materials were also demonstrated to be capable of producing an identical inhibitory effect. Finally, it was suggested that the application of recalcitrant >1K materials may represent a novel approach to the control of deleterious microbial communities.

Journal Article↗
PubMed1991

Glucuronate-2-sulphatase activity in cultured human skin fibroblast homogenates.

The optimization of the assay conditions to detect glucuronate-2-sulphatase (GS) activity present in cultured human skin fibroblast homogenates towards a heparin-derived disaccharide substrate O-(beta-D-glucuronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[l-3H]mannitol 6-sulphate (GSMS) has shown that a complex relationship exists between pH, buffer composition, ionic strength and the influence of added BSA and salts (NaCl, Na2SO4, CuCl2 and ZnCl2) to achieve maximum sulphatase activity. Whereas albumin stimulated GS activity by more than 2-fold over the pH range 2.7-5.7, CuCl2 stimulated GS activity over the narrow pH range 3.0-4.2, and inhibited GS activity at higher pH. ZnCl2 stimulated GS activity more than 3-fold at pH 3.0 and by more than 10-fold at pH 4.8. NaCl inhibited GS activity at pH 3.0, while activity between pH 4.2 and 4.8 was stimulated by up to 10-fold, resulting in a shift in the observed pH optimum from 3.0 to 4.8 in the presence of 315 mM-NaCl. Skin fibroblast GS activity toward GSMS had apparent Km values of 0.5-1.2 microM at pH 3.0, and 27.0-33.2 microM at pH 4.8. Albumin stimulated GS activity at both low and high pH by an increase in the apparent Vmax. values without significant alteration in the respective Km values. At pH 4.8, NaCl stimulated GS activity as a result of an increase in Vmax. values. These observations raise the possibility that two forms of GS activity are present in skin fibroblast homogenates: a low-Km form that has a pH optimum of 3.0 and is stimulated by BSA and a high-Km form with a pH optimum of 4.8 which is stimulated by NaCl.

Buffers↗
PubMed1991

Sanfilippo D syndrome: correction of glucosamine-6-sulphatase deficiency following fibroblast culture in Chang's media.

The de-O-sulphation of alpha-linked glucosamine-6-sulphate residues in heparan sulphate requires a specific sulphatase, glucosamine-6-sulphatase, which has been shown to be deficient in tissues of Sanfilippo D, or mucopolysaccharidosis type IIID (MPS IIID), patients. MPS IIID fibroblasts cultured in Basal Eagle's medium supplemented with either fetal calf serum or heat-inactivated fetal calf serum, MDCB or Ultraserg media had residual glucosamine-6-sulphatase activities towards a heparin-derived trisaccharide substrate, O-(alpha-N-acetylglucosamine-6-sulphate)-(1----4)-L-O-(alpha- iduronic acid-2-sulphate)-(1----4)-D-O-2,5-anhydro[1-3H]mannitol-6- sulphate, GlcNAc6S-IdoA2S-anM6S, which were less than 1 per cent of the normal range for fibroblasts cultured in Basal Eagle's medium supplemented with fetal calf serum. However, the glucosamine-6-sulphatase activities of MPS IIID fibroblasts grown in Chang's medium were similar to the activities in normal control fibroblasts which were cultured in Basal Eagle's medium. These results indicate that caution is required for prenatal diagnosis of MPS IIID patients using chorionic villi or amniotic cells cultured in Chang's medium.

Cells, Cultured↗
PubMed1991

Sanfilippo syndrome type D in two adolescent sisters.

We report on two adolescent sisters with Sanfilippo syndrome type D with some clinical features different from other cases previously described. They are the oldest cases reported to date and provide new clues about the course of the disease. Enzymatic and immunological characterisation of the patients' fibroblasts indicated deficiency of N-acetylglucosamine-6-sulphate sulphatase (GlcNAc-6S sulphatase). However, Northern blot analysis showed apparently normal mRNA encoding GlcNAc-6S sulphatase. These findings suggest that abnormal translation or premature degradation may be responsible for the enzyme defect in these cases of Sanfilippo syndrome type D.

Adolescent↗
PubMed1990

Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties.

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

Carbohydrate Sequence↗
PubMed1989

Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties.

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.

Catalysis↗
PubMed1989

Sanfilippo D syndrome: estimation of N-acetylglucosamine-6-sulfatase activity with a radiolabeled monosulfated disaccharide substrate.

N-Acetylglucosamine-6-sulfatase activity was assayed by incubation of the radiolabeled disaccharide O-(a-N-acetylglucosamine-6-sulfate)-(1----3)-L-[6-3H]-idonic acid (GlcNAc6S-IdOA), with homogenates of leucocytes, cultured fibroblasts, and urine from normal individuals, patients affected with N-acetylglucosamine-6-sulfatase-deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses and lysosomal storage disorders. The assay clearly distinguished affected homozygotes from their obligate heterozygotes and normal controls and other lysosomal storage disorders. Sulfatase activity in fibroblasts, leucocytes, and urine toward GlcNAc6S-IdOA exhibited a pH optimum at 4.2, 4.5, and 5.1, respectively. Sulfatase activity in fibroblasts had an apparent Km of 7.2 microM and was significantly inhibited by both sulfate and phosphate ions. The action of fibroblast or leucocyte N-acetylglucosamine-6-sulfatase activity toward GlcNAc6S-IdOA is recommended for the routine enzymatic detection and classification of mucopolysaccharidosis type IIID patients.

Amnion↗
PubMed1989

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria.

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.

Bacteriological Techniques↗
PubMed1988

Human glucosamine-6-sulfatase cDNA reveals homology with steroid sulfatase.

Glucosamine-6-sulfatase is a lysosomal enzyme which degrades glycosaminoglycans and is deficient in mucopolysaccharidosis type IIID. Human liver contains two major active forms of glucosamine-6-sulfatase, form A which has a single 78 kDa polypeptide and form B which has two polypeptides of 48 kDa and 32 kDa. A 1761 base pair cDNA clone encoding the complete 48 kDa polypeptide of form B was isolated. Form A is shown to be processed to form B with the 48 kDa polypeptide C-terminal to the 32 kDa polypeptide, and it is shown that C-terminal processing is limited to a region of thirty amino acids. The glucosamine-6-sulfatase sequence reveals homology with steroid sulfatase, a microsomal enzyme.

Amino Acid Sequence↗
PubMed1988

Early assessment of the effect of map-guided transcatheter intracardiac electric shock on sustained ventricular tachycardia secondary to coronary artery disease.

Short-term efficacy of transcatheter intracardiac electric shock was evaluated in 25 patients with recurrent sustained ventricular tachycardia (VT) secondary to coronary artery disease. Pace mapping and activation sequence mapping during VT were both used to direct intracardiac electric shocks applied between the distal electrode of the catheter (cathode) and a chest wall plate (anode) using a mean total energy of 588 +/- 209 J/patient. The short-term outcome of the procedure was considered successful in 3 patients (12%) who neither developed spontaneous VT nor had sustained VT in response to programmed electrical stimulation 1 week after the procedure. The short-term outcome in the remaining 22 patients (88%) was considered unsuccessful: 7 of the 22 patients developed spontaneous sustained VT and in 19 patients, including 15 with no spontaneous VT, sustained VT was still inducible by programmed electrical stimulation 7 +/- 2 days after the intracardiac electric shock. All 22 patients were subsequently treated with other modes of antiarrhythmic therapy. Using the technique described, the short-term efficacy of map-directed transcatheter intracardiac electric shock in eliminating VT is poor.

Adult↗
PubMed1988

The excretion of large vitamin C loads in young and elderly subjects: an ascorbic acid tolerance test.

An ascorbic acid tolerance test is described for assessing vitamin C status. The test is simple to administer and suitable for elderly patients. It involves giving an oral load of 1 g ascorbic acid in water and then measuring urinary excretion of vitamin C over the next 6 h. The excretion pattern at dosing has been studied in ten young subjects. The result of the ascorbic acid tolerance test in these young subjects was significantly different after supplementation with 1 g ascorbic acid daily for 1 month. Two series of elderly patients were also studied with the ascorbic acid tolerance test. They had low initial plasma ascorbic acid levels and much less vitamin C was excreted in the urine after dosing. Seven of these elderly patients were then supplemented with 1 g ascorbic acid for 1 month. After supplementation the initial plasma levels and their response to the ascorbic acid tolerance test became similar to that seen in younger subjects.

Administration, Oral↗
PubMed1987

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.

Chromatography, Liquid↗
PubMed1987

Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties.

Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.

Carbohydrate Metabolism↗