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S Gatt

Publications and source records attributed to S Gatt.

At least 19 recordsLinked to original sources
PubMed1992

Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.

Cell Survival↗
PubMed1992

Photosensitization of human bladder carcinoma cells by pyrene-dodecanoic acid: quantitative analysis of the cytotoxicity.

The effects of photochemotherapy with the fluorescent fatty acid pyrenedodecanoic acid (P12) and long-wavelength ultraviolet (UVA) light on cells derived from human bladder carcinoma were studied. Exposure of these anchorage-dependent cells to P12 either in monolayers of adherent cells or in suspension resulted in a time-related uptake of P12 and its incorporation into the cells' neutral and phospholipids. The uptake and localization of P12 was visualized with fluorescence microscopy and the distribution of the cell population with respect to P12 uptake was analyzed by flow cytometry. Irradiation of P12-containing monolayers of bladder carcinoma cells with UVA light resulted in cell killing. But, on microscopic examination no apparent cell lysis was detected, and since digestion with trypsin did not result in the dispersion of the monolayers it was impossible to assess toxicity by cell count. Alternative procedures were therefore used, and the following cell parameters were determined: (a) cellular uptake or release of chromate; (b) ability of cells to re-adhere to the substratum; and (c) the long-range proliferation potential. The combined inhibitory effect of photoirradiation on cell adherence and on their proliferative potential was utilized for determining reductions of up to 7 log in cell viability. The results obtained with five independently established in vitro bladder carcinoma cell lines indicated that these cells are susceptible to P12-induced photosensitization, suggesting that bladder malignancies might be potential candidates for pyrene-induced photochemotherapy.

Carcinoma↗
PubMed1992

Pyrene butanol--an efficient, selective and non-metabolized photosensitizing agent for human myeloid leukemia cells.

Methods for ex vivo purging of neoplastic cells from harvested marrow are being developed to increase the efficacy of autologous transplantation. One approach is selective photosensitization, using sensitizing compounds and light radiation. Pyrene-containing fatty acids and lipids are potent photosensitizers, e.g. 12-(1-pyrene)dodecanoic acid (P12), is taken up preferentially by leukemic cells and undergo photoexcitation when exposed to long wave ultra-violet light, resulting in selective killing of leukemic cells. These compounds are incorporated into the neutral- and phospho-lipids of the cells. The presence of intracellular pyrene-linked lipids might present a potential hazard in applying these agents for clinical use. We have, therefore, studied a series of other pyrene-linked compounds with the objective of finding a non-metabolizable photosensitizing agent that can be easily removed from the cells. In the present paper we report the results with pyrene butanol (P4-OH), a pyrene linked short-chain alcohol. When compared to P12, P4-OH was found to be taken up by cells most rapidly and reached saturation within minutes. It did not undergo any metabolism and washing the cells with serum-containing salt solutions removed practically all the P4-OH. This compound was found to be an efficient photosensitizer (in terms of concentrations and time of incubation with the cells) and selective to leukemic cells--it caused a 99% reduction in leukemic clonogenic cells under conditions that normal hemopoietic progenitors remained almost intact. These properties make P4-OH a potential photosensitizer for clinical application.

Acute Disease↗
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↗
PubMed1991

Different pathways of uptake and degradation of sphingomyelin by lymphoblastoid cells and the potential participation of the neutral sphingomyelinase.

The metabolism of sphingomyelin (SPM) was investigated in Epstein-Barr virus-transformed lymphoid cell lines from normal individuals and from patients with Niemann-Pick disease Type A (deficient in the acid, lysosomal sphingomyelinase) and familial hypercholesterolemia (lacking the low density lipoprotein receptor). Cells were incubated with the following radioactive or fluorescent SPMs: [choline-methyl-14C] SPM, [oleoyl-3H]SPM, pyrene-propenoyl-SPM (P3:1-SPM), pyrene-butanoyl-SPM (P4-SPM), pyrene-dodecanoyl-SPM (P12-SPM), and pyrene-sulfonylamino-undecanoyl-SPM (PSA11-SPM). Several pathways of uptake and subsequent metabolism of SPM in the lymphoblastoid cells were identified. [choline-methyl-14C]SPM and the P12-analog, administered to the cells in the presence of lipoproteins, were taken up through the apoB/E receptor-dependent pathway of endocytosis and degraded solely by the lysosomal sphingomyelinase. Under similar conditions, the other sphingomyelins, i.e. [oleoyl-3H]SPM, P3:1-SPM, P4-SPM, and PSA11-SPM, were taken up by a low density lipoprotein receptor-independent pathway and degraded mostly by a nonlysosomal sphingomyelinase which also catalyzed their hydrolysis in Niemann-Pick cells. In the absence of serum, all sphingomyelins were taken up by an apoB/E receptor-independent pathway and hydrolyzed by a nonlysosomal sphingomyelinase. Indeed, in vitro assays demonstrated the presence, in lymphoblastoid cells, of the neutral magnesium-activated sphingomyelinase, which was also fully active in the Niemann-Pick cells. In conclusion, our observations are consistent with: (i) the existence in lymphoblastoid cells of several pathways for the uptake and subsequent utilization of SPM; (ii) a major role of lipoproteins for the metabolic routing of the SPM; and (iii) the effect of the structure of the fatty acyl residue of SPM on its possible association with lipoproteins and/or cell membranes.

Blood↗
PubMed1991

Administration of pyrene lipids by receptor-mediated endocytosis and their degradation in skin fibroblasts.

Sphingomyelin and seven glycosphingolipids were labeled with the fluorescent probe pyrene and administered into cultured fibroblasts by receptor-mediated endocytosis. For this purpose pyrene sphingomyelin or mixtures of pyrene glycolipid and unlabeled sphingomyelin were dispersed as small, unilamellar liposomes. Apolipoprotein E was then added and the receptor for this ligand on the cell surface was utilized for uptake of the liposomes and their transport to the lysosomes, where the respective pyrene lipids were degraded. Following incubation with each of the respective pyrene lipids, only the administered compound and the pyrene ceramide were present; intermediate hydrolysis products were not detected. This indicated that, in skin fibroblasts, the lysosomal ceramidase was limiting and controlled the rate of total degradation of the pyrene sphingolipids.

Apolipoproteins E↗
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↗