Lysosomes disruption

Buffer components Tris 20 mM, pH7.4 maintain pH minimise acidification caused by lysosomal disruption... 

Some sensitizers, such as the water-soluble TPPS and AlPcS, localize in lysosomes, which disrupt during PDT [88,89]. The sensitizers are then relocalized in the cells and may even enter the nucleus [89]. Lysosomal damage leads to leakage of the contents of the lysosomes into the cytoplasm. One of the hottest and most promising applications of PDT may turn out to be photochemical internalization the use of PDT to liberate molecules taken up by endosomes and lysosomes (toxins, DNA fragments etc.) into the cytoplasm. Several extremely promising applications of this technique have been proposed and demonstrated [90] since lysosomal disruption in vivo had been demonstrated in 1996 [91]. 

Barn M, et al. (1998). Lysosome-disrupting peptide increases the efficiency of in-vivo gene transfer by liposome-encapsulated DNA. J. Drug Target. 6 191-199. 

Selden C, Owen M, Hopkins JMP, Peters TJ (1980) Studies on the concentration and intracellular localization of iron proteins in liver biopsy specimens from patients with iron overload with special reference to their role in lysosomal disruption. Br J Haematol 44 593-603... 

Van Rossenberg S.M. et al. Targeted lysosome disruptive elements for improvement of parenchymal liver cell-specific gene delivery, /. Biol Chem., 277,45803, 2002. 

C1C-6 is a late endosomal chloride transporter. Its disruption in mice led to lysosomal storage disease. C1C-7 is expressed in late endosomes and lysosomes. It needs Ostml as (3-subunit [3]. The disruption of either C1C-7 or Ostml in mice and man leads to severe osteopetrosis, retinal degeneration, and a severe lysosomal storage disease. ClC-7/Ostml is highly expressed in osteoclasts. In these cells, it is inserted together with the proton pump into the specialized plasma membrane ( ruffled border ) that faces the reabsorption lacuna. Osteoclasts are still present in C1C-7 knockout... 

Colchicine (a drug used in treatment of gout) and vinblastine (a cancer chemotherapy agent) may decrease liver uptake of americium. In rats that received an intraperitoneal injection of either colchicine and vinblastine prior to an intravenous or intramuscular injection of americium citrate, liver uptake of americium was lower, relative to controls, and kidney and skeletal americium uptake were higher (Seidel 1984, 1985). The effect is thought to involve disruption of hepatic microtubule formation, which is critical to the formation and intracellular processing of lysosomes, the initial site of accumulation of americium in the liver. 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

Brunk, U.T., Dalen, H., Roberg, K.., and Hellquist, H.B., 1997, Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts. Free Radio. 

Schneider, D.L. (1981). ATP-dependent acidification of intact and disrupted lysosomes Evidence for an ATP-driven proton pump. J. Biol. Chem. 256, 3858-3864. 

Many biological cells contain degradative enzymes (proteases) that catalyze the hydrolysis of peptide linkages. In the intact cell, functional proteins are protected from these destructive enzymes because the enzymes are stored in cell organelles (lysosomes, etc.) and released only when needed. The proteases are freed upon cell disruption and immediately begin to catalyze the degradation of protein material. This detrimental action can be slowed by the addition of specific protease inhibitors such as phenylmethyl-sulfonyl fluoride or certain bioactive peptides. These inhibitors are to be used with extreme caution because they are potentially toxic. 

After cell disruption, gross fractionation of the properly stabilized, crude cell homogenate may be achieved by physical methods, specifically centrifugation. Figure 7.11, Chapter 7, outlines the stepwise procedure commonly used to separate subcellular organelles such as nuclei, mitochondria, lysosomes, and microsomes. 

Oligomannosyl carbohydrates on soluble enzymes destined to become lysosomal enzymes carry one or two phosphate residues at the 6 position of mannose (Man-6-P). These phosphorylated mannose residues are recognized by a glycoprotein called the Man-6-P receptor, which binds and transports the prelysosomal enzymes to prelysosomal vesicles (see fig. 16.13). This binding ensures that the prelyso-somal enzyme enters a vesicle destined to fuse to and thereby deliver its contents to the lysosome. In an acidic, prelysosomal compartment the binding between the Man-6-P receptor and the lysosomal enzyme is disrupted so that the receptor can be recycled to the Golgi apparatus. 

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