Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Ultrastructural analysis of pathologic lesions in sterol-deficient Nippostrongylus brasiliensis larvae

A variety of cellular lesions were manifested by the free-living larval stages of Nippostrongylus brasiliensis cultured axenically in medium lacking cholesterol. Pathologic changes developed rapidly and were most apparent in intestinal cells which displayed generalized degradation of membranous organelles. Mitochondria, endoplasmic reticulum, and Golgi complexes became disassociated and vacuolated. Autophagosomes appeared within intestinal cells and contained a wide variety of cellular components. By the 5th day gross vacuolization and degeneration of intestinal cells occurred and the hypodermis and lateral cords displayed lysed cytoplasmic regions. The latter structures are concerned with synthesis of cuticle and their degeneration correlates with the suppression of molting and the abnormal molts that occurred.     

Kurloff cell lysosomal arylsulphatases: Presence of both cationic and highly anionic isoforms of the sole B class

Following the previous ultrastructural demonstration of the presence of arylsulphatase (Asase) activities in Kurloff cells (KC) and of their quasi-exclusive localization in the Kurloff body (KB), this work investigates their biochemical and zymographic properties after extraction from purified KC suspensions. Using the discriminative inhibitory conditions of both the Baum or LeeVaupel and Conzelmann methods, nitrocatechol sulphate hydrolyzing enzymes of the KC were assumed to belong to the B class of the type II Asase alone. After electrophoretic separation under non-denaturing conditions in a 4–23% polyacrylamide gel, they were characterized by 55 kDa and 62 kDa zymographic bands. After isoelectric focusing, ‘classical’ cationic isoforms (pI 8.5) and two anionic isoforms (pI 4.4 and 4.6) were observed on zymograms. As expected for class B Asase, the different zymographic forms of KC Asase were only recovered in the unadsorbed fraction after anion-exchange chromatography on DEAE-cellulose column equilibrated with high ionic strength buffer. Their K_m (2.1 mM), their optimum pH (5.8) and their inhibitions by sulfite, phosphate, sulphate and ascorbic acid as well as their slight stimulation by AgNO₃ were also characteristic of this class of Asase. Finally, chondroitin4-sulphate was shown to potentially be a physiological substrate for these lysosomal enzymes.     

Selective intra-lysosomal concentration of niobium in kidney and bone marrow cells: a microanalytical study

Niobium is used as an alloy in the industrial and biomedical fields. The concentration of the toxic element in organs of a number of animal species has been defined by using radioactive niobium (⁹⁵Nb). However, tissue lesions induced by niobium have only been studied at the light microscopy level. In this study, we used an electron probe X-ray analyzer equipped with a transmission electron microscope to define the localization of this element in kidney and bone marrow cells. Results demonstrated that niobium is located in the lysosome and that this element coprecipitates with phosphate. In kidney, lysosomes and precipitates are eliminated in the tubular lumen. In contrast, precipitates appear to be eliminated more slowly from the lysosomes of bone marrow macrophages. These processes therefore correspond to one of the mechanisms by which lysosomes eliminate certain toxic mineral elements and thus play a role in the more general process of the body's defenses.     

Lysosomal Membrane Proteins of Amoeba proteus, as Studied with Monoclonal Antibodies

Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomoes from fusing with them.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis

BACKGROUND: The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm. METHODS: The mechanism of PEI-mediated DNA transfer was investigated using quantitative methods to study individual steps in the overall transfection process. In addition to transfection efficiency, the cellular uptake, local pH environment, and stability of vectors were analyzed. N-Quaternized (and therefore non-proton sponge) versions of PEI and specific cell function inhibitors were used to further probe the proton sponge hypothesis. RESULTS: Both N-quaternization and the use of bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately two orders of magnitude. Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N-quaternized PEIs and polylysine by 2-3-fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Significantly lower average pH environments were observed for permethyl-PEI (pH 5.4), perethyl-PEI (pH 5.1), and polylysine (pH 4.6) vectors. Cellular uptake levels of permethyl-PEI and perethyl-PEI vectors were found to be 20 and 90% higher, respectively, than that of parent PEI vectors, indicating that the reduction in transfection activity of the N-quaternized PEIs is due to a barrier downstream of cellular uptake. A polycation/DNA-binding affinity assessment showed that the more charge dense N-quaternized PEIs bind DNA less tightly than PEI, demonstrating that poor vector unpackaging was not responsible for the reduced transfection activity of the N-quaternized PEIs. CONCLUSIONS: The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).