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Biomedical subjects

S Gatt

Publications and source records attributed to S Gatt.

At least 91 records · Page 5Linked to original sources
PubMed1986

Esmolol for control of increases in heart rate and blood pressure during tracheal intubation after thiopentone and succinylcholine.

Esmolol, an ultra-short-acting cardioselective beta-adrenergic blocker, was investigated in a double-blind prospective protocol for its ability to control haemodynamic responses associated with tracheal intubation after thiopentone and succinylcholine. Thirty ASA physical status I patients received a 12-minute infusion of esmolol (500 micrograms X kg-1 X min-1 for four minutes, then 300 micrograms X kg-1 X min-1 for 8 minutes) or saline. Five minutes after the start of the drug/placebo infusion, anaesthesia was induced with 4 mg X kg-1 thiopentone followed by succinylcholine for tracheal intubation. Prior to induction esmolol produced significant decreases in heart rate (HR) (9.3 +/- 1.8 per cent) and rate-pressure product (RPP) (13.1 +/- 1.8 per cent), systolic blood pressure (SAP) (4.3 +/- 1.5 per cent) and mean arterial blood pressure (MAP) (1.7 +/- 2.0 per cent). Increases in HR, SAP and RPP after intubation were approximately 50 per cent less in patients given esmolol compared to patients given placebo. There were highly significant differences in HR (p less than 0.0001), and RPP (p less than 0.0005) and significant differences in SAP (p less than 0.05) when the maximal esmolol post-intubation response was compared to the maximal placebo response. Infusion of esmolol in the dose utilized in this study significantly attenuated but did not completely eliminate cardiovascular responses to intubation.

Adult↗
PubMed1986

Uptake of fluorescent fatty acids by erythroleukemia cells. Effect of differentiation.

The uptake of a fluorescent derivative of a fatty acid (FDFA), 12-(1-pyrene) dodecanoic acid (P12) by murine erythroleukemia (MEL) cells was studied. Because of the intense fluorescence of the pyrene ring, the association of P12 with intact cells could be analysed using a fluorescence microscope or a fluorescence-activated cell sorter (FACS), and the incorporation of P12 into cellular lipids could be quantified, following their extraction in a spectrofluorimeter. These procedures indicated that P12 uptake and intracellular utilization are reduced, following induction of erythrodifferentiation by dimethylsulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA). The differences in the fluorescence observed following exposure to P12 permitted us to separate a mixture of differentiated and undifferentiated cells into two distinct cell subpopulations; the high fluorescence population consisted mainly of undifferentiated cells, and the low one of differentiated cells. The results of this study suggest that fluorescent fatty acids are useful for distinguishing between and sorting cells at different stages of differentiation.

Acetamides↗
PubMed1986

Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site.

Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3H-labeled conduritol B epoxide (3H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V8 protease digestion of the 3H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (Mr, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid beta-glucosidase.

Amino Acid Sequence↗
PubMed1986

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

1-Deoxynojirimycin↗
PubMed1986

Photosensitization to ultraviolet irradiation and selective killing of cells following uptake of pyrene-containing fatty acid.

Cells were incubated with 12-(1-pyrene)-dodecanoic acid (P12), a long-chain fatty acid to which a pyrene ring has been attached covalently. This acid was transported across the plasma membranes of cells and subsequently incorporated into their neutral lipids and phospholipids. Irradiation of these pyrene-containing cells for short periods (0.5-4 min) with ultraviolet light at 366 nm resulted in eventual cell death. Similar irradiation had no effect on cells that had not been exposed to P12. The time of the period of irradiation necessary for inducing the toxic process was related to the quantity of P12 incorporated, the latter being a function of the respective metabolic activity of the individual cell type. The degree of incorporation of P12 into a cell, and consequently its acquired sensitivity to killing by ultraviolet irradiation at 366 nm, was affected by the incubation temperature and addition of non-fluorescent fatty acid, albumin or other serum proteins. Different degrees of incorporation of P12 into various cell types were used for selective killing and elimination of cell populations by irradiation at 366 nm. The combined procedure of preincubation with P12 followed by ultraviolet irradiation thus permitted selection of cell types with a greater resistance to this procedure.

Cell Line↗
PubMed1985

Genetic heterogeneity in Gaucher disease: physicokinetic and immunologic studies of the residual enzyme in cultured fibroblasts from non-neuronopathic and neuronopathic patients.

To elucidate the genetic heterogeneity in the three major phenotypic subtypes of Gaucher disease, the residual acid beta-glucosidase in fibroblasts from patients with all three subtypes from different ethnic and demographic groups was investigated by comparative kinetic, thermostability, and immunotitration studies. The kinetic studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to the enzyme modifiers, taurocholate (or phosphatidylserine), and glucosyl sphingosine (or N-hexyl glucosyl sphingosine); Group A residual enzymes responded normally to these modifiers. All neuronopathic patients (types 2 and 3) and most non-Jewish, non-neuronopathic patients (type 1) had group A residual activities and thus could not be distinguished by their kinetic properties. Group B residual enzymes had markedly abnormal responses to these modifiers. All Ashkenazi and only two non-Jewish type 1 patients had group B residual activities. Group C residual activity had an intermediate response to all modifiers and represented a single Afrikaner type 1 patient. Pedigree studies indicated that this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. Thermostability studies showed additional heterogeneity of the residual activities within the three kinetic groups. Group A (type 2) and group B (type 1) enzymes had similarly decreased thermostabilities. In contrast, group A (type 1) residual activities were heterogeneous; three classes of thermostabilities were found among these enzymes: normal, decreased, and increased. Immunotitration of equal amounts of the normal or Gaucher disease beta-glucosidase activities with monospecific IgG indicated that the enzyme proteins from most Gaucher disease patients were antigenically altered and/or that large amounts of catalytically abnormal or inactive antigen were present. A decreased amount of antigenically and catalytically normal enzyme was present in a group A, type 1 African black patient, suggesting decreased stability or synthesis of his mutant acid beta-glucosidase. These kinetic, immunologic, and thermostability studies indicated that 1) type 1 Gaucher disease is biochemically heterogeneous and results from at least four distinct allelic acid beta-glucosidase mutations that alter enzyme structure and/or function, 2) neuronopathic and non-Jewish non-neuronopathic phenotypes cannot be distinguished reliably by kinetic analyses alone, and 3) the Ashkenazi type 1 Gaucher disease results from a unique mutation that alters a specific active site domain of acid beta-glucosidase.

Cells, Cultured↗
PubMed1985

New spectrophotometric assays of acid lipase and their use in the diagnosis of Wolman and cholesteryl ester storage diseases.

Trinitrophenylaminolauric acid (TNPAL) was linked to glycerol or cholesterol and the resulting yellow compounds were used as substrates for several lipases and cholesteryl esterase in cells from normal individuals and patients with Wolman's or cholesteryl ester storage diseases. Normal cells (lymphoid cell lines or skin fibroblasts) showed two peaks of lipase or cholesteryl esterase activity at about pH 4.0 and 6.0 each. The activity of the most acidic enzyme, which hydrolyzed natural or synthetic triacylglycerols as well as cholesteryl esters, was considerably reduced in cells derived from patients with Wolman's or cholesteryl ester storage diseases. Simple spectrophotometric procedures were developed for using tri-TNPAL glycerol or TNPAL cholesterol to identify homozygotes of these two respective diseases.

Adult↗
PubMed1985

Use of activators and inhibitors to define the properties of the active site of normal and Gaucher disease lysosomal beta-glucosidase.

Lysosomal beta-glucosidase ('glucocerebrosidase') in peripheral blood lymphocyte and spleen extracts from normal individuals and Ashkenazi-Jewish Gaucher disease type-1 patients were investigated using several modifiers of glucosyl ceramide hydrolysis. The negatively charged lipids, phosphatidylserine and taurocholate, had differential effects on the hydrolytic rates of the normal and Gaucher disease enzymes from either source. With the normal enzyme, either negatively charged lipid (up to 1 mmol/l) increased the reaction rates, while decreasing hydrolytic rates were obtained at greater concentrations. In comparison, the peak activities of the Gaucher enzymes were observed at about 2-3 mmol/l or 5-8 mmol/l of phosphatidylserine or taurocholate, respectively. These negatively charged lipids altered only the velocity of the reactions; the apparent Km values were not affected. Taurocholate or phosphatidylserine also facilitated the interaction of the normal enzyme with conduritol B epoxide, a covalent inhibitor of the catalytic site. Compared to the normal enzyme, the Ashkenazi-Jewish Gaucher type-1 enzyme required about 5-fold greater concentrations of conduritol B epoxide for 50% inhibition. Neutral or cationic acyl-beta-glucosides were found to be competitive or noncompetitive inhibitors of the enzymes, respectively. Alkyl beta-glucosides were competitive (or linear-mixed type) inhibitors of the normal splenic or lymphocyte enzyme with competitive inhibition constants (Ki) inversely related to the chain length. With octyl and dodecyl beta-glucoside nearly normal competitive Ki values were obtained with the splenic enzymes from Gaucher patients. These Ki values were not influenced by increasing phosphatidylserine or taurocholate concentrations. In contrast, the cationic lipids, sphingosyl-1-O-beta-D-glucoside (glucosyl sphingosine) and its N-hexyl derivative, were noncompetitive inhibitors whose apparent Ki values for the normal enzyme were 30 and 0.25 mumol/l, respectively. The Ki values for these sphingosyl glucosides were about increased 5 times for the Gaucher type-1 enzymes from Ashkenazi-Jewish Gaucher disease type-1 patients. The Ki values of glucosyl sphingosine for the normal or mutant enzymes were directly related to increasing concentrations of phosphatidylserine or taurocholate. This latter site appears to be specifically altered by a mutation in the structural gene for lysosomal beta-glucosidase in the Ashkenazi-Jewish form of type-1 Gaucher disease.

Binding Sites↗
PubMed1985

Use of mixed dispersion of fluorescent galactosylceramide and sodium dodecylsulfate for assaying galactosylceramide-beta-galactosidase and diagnosing Krabbe disease.

A fluorescent derivative of galactosylceramide [NBD-GalCer: 12-N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminodecanoyl+ ++ galactosylceramide] was dispersed in sodium dodecylsulfate and used to determine the activity of galactosylceramide-beta-galactosidase. Optimal assay conditions were defined, compared to other assay procedures and found suitable for diagnosing Krabbe disease.

4-Chloro-7-nitrobenzofurazan↗
PubMed1985

Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.

To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax.

Adolescent↗
PubMed1984

Prenatal diagnosis of Krabbe disease using a fluorescent derivative of galactosylceramide.

A fluorescent substrate 12-(N-methyl-N(7-nitro-2-oxa-1,3-diazol-4-yl) aminododecanoyl sphingosyl beta-D-galactoside ('NBD galactocerebroside') was synthesized and used for the detection of galactocerebrosidase activity. The enzyme determinations using this substrate were found to be extremely sensitive yielding unambiguous results. This substrate was used for the prenatal diagnosis of a fetus affected with Krabbe disease; the diagnosis was later confirmed in the aborted fetus.

Cells, Cultured↗
PubMed1984

Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites.

Three binding sites on highly purified lysosomal beta-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-beta-D-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (less than 1.8 mM) or phosphatidylserine (0.5 mM). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and its N-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-beta-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-beta-D-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl beta-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki (slope)] for the two alkyl beta-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki (intercept)] was nearly identical for both alkyl beta-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl beta-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal beta-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the beta-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.

Binding Sites↗
PubMed1984

Induction of lipid storage in cultured leukemic myeloid cells by pyrene-dodecanoic acid.

When incubated for 1-3 days in the presence of the fatty acid analog, 12-(1-pyrene)dodecanoic acid, the neutral lipid content of cultured human leukemic myeloid cells increased considerably, while that of the phospholipids increased to a much lesser extent. Among the neutral lipids, di- and monoacylglycerols predominated and a considerable portion of the fatty acyl residues of these newly synthesized neutral lipids consisted of pyrene-dodecanoic acid. Light microscopy showed evidence for the presence of highly fluorescent lipid droplets within the cells. Electron microscopy showed lipid globules, mostly devoid of a unit membrane, multivesicular inclusion bodies and some multilamellar membranous structures. In comparison, cells incubated with palmitic acid show neither these cellular structures, nor the increase of the neutral lipid content. The lipid storage, induced by pyrene-dodecanoic acid, is probably related to ineffective degradation of this fatty acid analog and might serve as an experimental model of cellular lipidosis.

Cell Division↗
PubMed1984

Utilization of membranous lipid substrates by membrane-bound enzymes. Intramembrane and intermembrane hydrolysis of diacylglycerol by lipase of rat liver microsomes.

The membranous lipase of rat liver microsomes was used to hydrolyze diacylglycerol (DG), generated within the microsomal membrane by treatment with phospholipase C, in two separate interactions. For an intramembrane enzyme-substrate interaction, the enzyme and DG were present in the same microsomes. For intermembrane interactions, native microsomes of rat liver were used as carriers of the enzyme, while heated and phospholipase C-treated microsomes of rat liver or brain were employed as carriers of the substrate. The v vs S curves of the intermembrane interaction were hyperbolic while those of the intramembrane utilization were parabolic.

Animals↗
PubMed1984

Synthesis of a fluorescent derivative of glucosyl ceramide for the sensitive determination of glucocerebrosidase activity.

A fluorescent derivative of glucosyl ceramide was synthesized by covalently linking a fluorescent fatty acid, 12-[N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminododecanoic acid to the amino group of sphingosyl-1-O-beta-D-glucoside, glucosyl sphingosine. For hydrolysis by glucocerebrosidase, this substrate was dispersed in mixed micelles with Triton X-100 and sodium taurocholate or in unilamellar liposomes with phosphatidylcholine and the negatively charged lipid, dicetylphosphate. In either micellar or liposomal dispersions of the fluorescent substrate, reaction rates were linear with time and protein concentration, and saturation kinetics were observed. The rate of hydrolysis of this fluorescent substrate was equal to that obtained with radiolabeled glucosyl ceramide. The fluorescent glucosyl ceramide was used to determine glucocerebrosidase activity in extracts of human leukocytes, cultured skin fibroblasts, and various tissues as well as in partially purified splenic and placental glucocerebrosidase preparations. This fluorescent derivative of the natural substrate was not hydrolyzed by aryl beta-glucosidase(s), thereby facilitating the specific and reliable diagnosis of heterozygotes and homozygotes with Gaucher disease.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Preparation and characterization of well defined D-erythro sphingomyelins.

A simple semisynthetic procedure for the preparation of various D-erythro sphingomyelins (SPMs), differing in their acyl chains, is described. They were prepared by one-step condensation of the desired free fatty acid with sphingosyl phosphorylcholine (SPC) using dicyclohexylcarbodiimide. The D-erythro SPMs were obtained in high purity, high yields and resemble bovine brain SPM in their chromatographic behavior, infrared, circular dichroism (CD) and proton NMR (PMR) spectra as well as in their rate of hydrolysis by Staphylococcus aureus sphingomyelinase. Multilamellar vesicles can be prepared from the semisynthetic SPMs. Their thermotropic behavior is dependent mainly on the acyl chain though it is also affected by the heterogeneity of the sphingosine base composition. Intact sealed small unilamellar vesicles (SUV) cannot be prepared from a single semisynthetic saturated SPM but can be prepared from their mixtures. This acylation procedure can also be applied for preparing simple neutral glycosphingolipids. The sphingolipids prepared by this method can be used to study metabolism, enzymology and physicochemical properties of D-erythro well defined simple sphingolipids.

Animals↗
PubMed1984

Lysosomal beta-glucosidase of rat liver.

Studies were undertaken to characterize the beta-glucosidase activity in freshly homogenized liver from Sprague-Dawley rats. About 95% of the total beta-glucosidase activity was associated with the particulate fraction, whereas only about 3-7% was found in the cytosol. Storage of fresh liver at room temperature for several hours or repeated freezing and thawing of fresh rat liver prior to homogenization, solubilized 20-30% of the total hepatic beta-glucosidase activity. An additional 30% could be solubilized by extracting the particulate sediments with water or Triton X-100. The enzymatic activity in both the particulate and solubilized fractions optimally hydrolyzed 4-methylumbelliferyl-beta-D-glucoside as well as the glycolipid substrate, glucosylceramide, at an acidic pH. The rates of hydrolysis of either substrate by all subcellular fractions were stimulated by addition of sodium taurocholate or phosphatidylserine. The particulate, cytosolic and solubilized enzymes bound to concanavalin A, were inhibited by conduritol B epoxide and migrated more electronegatively on cellulose acetate than the cytosolic acid beta-glucosidase from human liver or spleen. These data indicated that the liver of Sprague-Dawley rats contained primarily the lysosomal acid beta-glucosidase ('glucocerebrosidase') and little, if any, 'nonspecific' beta-glucosidase. This, and the fact that about 60% of the rat hepatic beta-glucosidase could be solubilized by autolysis, freezing and rethawing or extraction with water, contrasts with the beta-glucosidases in human liver since about 80% of the total beta-glucosidase activity is cytosolic and does not hydrolyze glucosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗