Search PubMed⌕ Search

Biomedical subjects

S Gatt

Publications and source records attributed to S Gatt.

At least 19 recordsLinked to original sources
PubMed1992

Toward gene therapy for Niemann-Pick disease (NPD): separation of retrovirally corrected and noncorrected NPD fibroblasts using a novel fluorescent sphingomyelin.

The neurologic (type A) and nonneurologic (type B) forms of Niemann-Pick disease (NPD) both result from deficiencies of acid sphingomyelinase (ASM) activity leading to the accumulation of sphingomyelin and other related lipids within lysosomes. Recently, the full-length cDNA and genomic sequences encoding ASM have been isolated and the nature of the molecular lesions causing NPD has been investigated. Although these developments have facilitated diagnosis for this debilitating disease, no effective treatment is currently available. Toward this latter goal, our laboratories recently reported the effectiveness of retroviral-mediated gene transfer for the in vitro correction of the cellular pathology in NPD fibroblasts (Suchi et al., 1992). In addition, novel selection procedures were developed to separate retrovirally corrected and noncorrected NPD fibroblasts based on the receptor-mediated delivery of a fluorescently (pyrene)-labeled sphingomyelin (P12-SPM) to the lysosomes of cells using liposomes coated with apolipoprotein E. In this study, we have used a different, fluorescent derivative of sphingomyelin (lissamine-rhodamine dodecanoyl sphingomyelin; LR12-SPM) to extend and improve this selection system. LR12-SPM offers a number of advantages over P12-SPM, including the facts that apolipoprotein E is not required for its efficient uptake and targeting to lysosomes and that the product of LR12-SPM degradation by ASM is efficiently transported out of cells. Thus, when analyzed in a fluorescence-activated cell sorter (FACS), there was complete separation (i.e., no overlap) of retrovirally corrected and noncorrected NPD cells after the administration of LR12-SPM.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Separation↗
PubMed1992

Photosensitization by Photofrin II delivered to WI26VA4 SV40-transformed human fibroblasts by low density lipoproteins: inhibition of lipid synthesis and fatty acid uptake.

Irradiation with 365 nm light of Wi26VA4 SV40-transformed human fibroblasts cultured for 24 h in the presence of low density lipoproteins loaded with the anticancer porphyrin mixture Photofrin II resulted in a near complete inhibition of [14C]oleic acid incorporation into triacylglycerols, cholesteryl esters and phospholipids. More than 80% reduction of the fatty acid incorporation in all lipid classes was observed following an irradiation dose of 1 J/cm2. The activities of the respective acyltransferases, measured in vitro on cell homogenates, were also markedly diminished, but to a lesser extent than lipid synthesis from oleic acid. Moreover, oleic acid uptake by cells was strongly and rapidly reduced. It is suggested that the rapid inhibition of membrane phospholipid synthesis upon cell photosensitization, due to both a direct inactivation of acyltransferases and to a reduction of fatty acid utilization, could play an important role in the photocytotoxic effect of Photofrin II.

Cell Line, Transformed↗
PubMed1992

A novel fluorescent pH indicator for the acidic range.

Reaction of a commercial preparation of lissamine-rhodamine B sulfonyl chloride with primary amines gives rise to two sulfonamide derivatives, of which one behaves as a pH indicator with maximal fluorescence emission in the acidic range and a pKa of about 4.6. The structure of this derivative and the mechanism of fluorescence dependence on pH, elucidated by means of NMR, is presented.

Acids↗
PubMed1979

Interaction of membranous enzymes with membranous lipid substrates. Hydrolysis of diacylglycerol by lipase in rat brain microsomes.

The phospholipids in rat brain microsomes were labeled with tritium by intracerebral administration of radioactive fatty acids and converted to diacylglycerol with phospholipase C. The latter lipid was hydrolyzed in situ at pH 4.8, to monoacylglycerol and fatty acid by the endogenous microsomal lipase. This paper provides an experimental approach to determine whether the lipid was degraded by enzyme molecules residing in its own membrane (intramembrane interaction) or an adjacent membrane (intermembrane interaction). Direct interaction between separate membranes containing enzyme or substrate showed the existence of the inter-membrane route while dilution experiments provided evidence for the presence of the intramembrane interaction as well. A probable difference in the mechanisms of these two interactions is suggested by different shapes of the curves that describe the reaction rate as a function of the endogenous substrate. The curve resulting from the intermembrane interaction was hyperbolic while that representing the intramembrane route was of a parabola-like shape. Competition experiments suggested that when given a choice between the two, the enzyme utilized preferentially the substrate molecules in its own membrane.

Animals↗
PubMed1978

Magnesium-dependent sphingomyelinase of infantile brain. Effect of detergents and a heat-stable factor.

The properties of the Mg2+-dependent sphingomyelinase, whose pH optimum is between 7 and 8, were investigated using post-mortem infantile brain. The enzyme could be extracted with 0.2% Triton X-100 and remained soluble when centrifuged at 170,000 X g. Subsequent removal of the detergent with SM2-Biobeads resulted in resedimentation of the enzyme at 80,000 X g. A detergent was needed for assaying enzymatic activity; either Triton X-100 or bile salts could be used. With increasing concentrations of detergent, the rates of hydrolysis of sphinomyelin increased, reached an optimum and then decreased, suggesting inhibition of the enzyme. The concentrations of detergent which resulted in optimal reaction rates were directly related to the protein concentration of the enzymatic preparation. A heat-stable factor which counteracts inhibition by the above detergents is present in brain as well as several other tissues. A lipid extract of the enzymatic preparation, or several purified lipids could not mimic the effect of the heat-stable factor. The interrelationship between enzyme, detergent and the heat-stable factor was investigated.

Brain↗
PubMed1978

Synthesis of trinitrophenylaminolauric acid and the use of its glyceryl esters for assaying lipase by a spectrophotometric procedure.

Trinitrophenylaminolauric acid was synthesized from omega-aminolauric acid and trinitrobenzenesulfonic acid and then condensed with glycerol to yield mono-, di- and triacylglyceryl esters of this acid. Hydrolysis of these glycerides was followed by isolation of the yellow fatty acid with the aid of one solvent extraction step and estimating its content by spectrophotometry. This procedure was used to assay the activities of lipases from hog pancreas, rat bile, microsomes of rat brain and Rhizopus arrhizus delamar.

Animals↗
PubMed1978

A spectrophotometric method for determination of sphingomyelinase.

A colored derivative of sphingomyelin was synthesized and used as substrate for several sphingomyelinases. The compound is N-omega-trinitrophenyl-aminolaurylsphingosylphosphorylcholine. The rate of hydrolysis of this substrate was compared to that of bovine brain sphingomyelin, labelled with tritium in the choline moiety. The following enzyme preparations were used: homogenate-less debris of brain, assayed at pH 5.0 or 7.4; a solubilized preparation derived from rat brain lysosomes, assayed at pH 5.0 and a purified enzyme of Staphylococcus aureus. With all preparations, the rates of hydrolysis of the yellow derivative were very similar to those of the brain sphingomyelin. Extracts of skin fibroblasts of normal and Niemann-Pick patients as well as amniotic cells were also used. Again, the rates of hydrolysis of the yellow derivative practically equalled those using brain sphingomyelin.

Animals↗
PubMed1978

The hydrolysis of triacylglycerol and diacylglycerol by a rat brain microsomal lipase with an acidic pH optimum.

Lipase activity towards triacylglycerol and diacylglycerol was measured at pH 4.8 using a microsomal preparation from rat brain as the enzyme source. The optimal pH for the hydrolysis of triacylglycerol was 4.8, with only minor lipolytic activity in the alkaline pH range. Diacylglycerol was the major product of triacylglycerol hydrolysis, with only little monoacylglycerol being formed. When diacylglycerol was the starting substrate it was hydrolyzed at a rate 10-fold greater than triacylglycerol, and the product was monoacylglycerol. The enzyme showed positional specificity for the fatty acid moieties located at the primary positions of sn-glycerol. 1,3-Diacylglycerol was hydrolyzed at greater than twice the rate of the corresponding 1,2(2,3)-isomer.

Animals↗
PubMed1977

Rate equations and simulation curves for enzymatic reactions which utilize lipids as substrates. I. Interaction of enzymes with the monomers and micelles of soluble, amphiphilic lipids.

Theoretical aspects of the kinetics of interaction of enzymes with lipid substrates are presented. Rate equations were written and used to simulate v versus S curves for interaction of enzymes with "monomers" (i.e. a molecular solution) or micelles (aggregated form) of the "soluble", amphiphilic lipids. The rate equations were written assuming separate kinetic parameters for the interaction of the enzyme with these two forms. Although the rate equations are based on the kinetic theory of Michaelis and Menten, most of the simulated v vs. S curves were not hyperbolic. A procedure is suggested for determining the kinetic parameters with the aid of a graphic method.

Colloids↗
PubMed1977

Rate equations and simulation curves for enzymatic reactions which utilize lipids as substrates. II. Effect of adsorption of the substrate or enzyme on the steady-state kinetics.

Theoretical aspects of the kinetics of interaction of enzymes with lipid substrates are presented. Rate equations were written and used to simulate v versus S curves for the following cases: (a) The substrate is adsorbed onto non-catalytic sites of the enzyme or to other proteins accompanying the enzyme. (b) The enzyme is adsorbed, via non-catalytic sites to aggregated forms of the substrate. (c) The substrate is adsorbed onto an externally added protein such as albumin. Although all rate equations are based on the Michaelis-Menten kinetic theory, most of the simulated v vs. S curves were not hyperbolic and some of the v vs. E curves not linear.

Adsorption↗
PubMed1976

Effect of Triton X-100 on the hydrolysis of sphingomyelin by sphingomyelinase of rat brain.

Mixed dispersions of the nonionic detergent Triton X-100 and sphingomyelin were used as substrate for sphingomyelinase of rat brain. The dependence of the rate of hydrolysis on the concentration of sphingomyelin was measured in two ways: at a fixed concentration of Triton X-100 or at varying concentrations of this detergent, while maintaining a fixed molar ratio of Triton X-100 to sphingomyelin. In either case, the upsilon vs. S curves deviated from the hyperbolic shape predicted by the Michaelis-Menten kinetic theory. These deviations are discussed and interpreted on the basis of the physicochemical properties of the mixed dispersions of detergent and lipid studied in previous papers.

Animals↗