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We found a dipeptidyl aminopeptidase activity in the parasitic protozoan Giardia lamblia with properties similar to the lysosomal cathepsin C of rat-liver lysosomes. Subcellular fractionation of this parasite indicated that the cathepsin C activity is located in organelles not distinguishable from the ones containing acid phosphatase, a known marker enzyme of Giardia lysosome-like peripheral vesicles. Contrary to the rat lysosomal enzyme, Giardia cathepsin C behaved like a membrane protein. Moreover, the enzyme was not solubilized by Triton X-100 or Triton X-100/SDS at 0 degrees C but could be substantially solubilized by octylglucoside, Triton X-100 at 37 degrees C or by a pretreatment with the cholesterol complexing agent beta-cyclodextrin before the Triton/SDS treatment carried out at 0 degrees C. These observations suggest that binding/anchorage of this enzyme to membranes occurs in cholesterol-rich microdomains.

Background: An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (hydrodynamics-based transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA., Methods: We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes)., Results: (35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face., Conclusions: The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA., (Copyright 2002 John Wiley & Sons, Ltd.)

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.

The cholesterol complexing agent methyl-cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl-L-phenylalanine 2-naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

Branched polyamines are extensively used as nonviral vectors for plasmid DNA in transfection experiments. Moreover, recently it has been shown that these compounds are able to eliminate prions from infected cells in cultures. It has been proposed that in both cases endosomes or lysosomes are the site of action. This raises the question of how these molecules are taken up by the cells and what is their intracellular fate. In the work presented here, the question has been addressed by investigating the uptake and the intracellular distribution of branched polyethyleneimine (25 kD) by centrifugation methods. The polyamine was labelled with (125)I-tyramine cellobiose and injected to the rat. The radioactive polymer is taken up after injection into the liver, kidney, spleen, and lungs and remains in these organs for many days. In the liver, it is found mainly in the hepatocytes. Intracellular distribution of radioactivity present in that organ was investigated by differential and isopycnic centrifugations. Early after injection, radioactivity exhibits a distribution pattern similar to that of alkaline phosphodiesterase, a plasma membrane marker. Later, the distribution pattern becomes similar to that of cathepsin C, a lysosomal enzyme. Radioactivity and hydrolase distributions in a sucrose gradient are similarly modified by a pretreatment of the rat with Triton-WR1339, a specific density perturbant of lysosomes. These results indicate that polyethyleneimine is endocytosed and reaches lysosomes. For many days it persists in these organelles probably due to its resistance to lysosomal hydrolases., (Copyright 2000 Academic Press.)

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.

Efficiency of transfection is probably dependent on the rate of intracellular degradation of plasmid DNA. When a non-viral vector is used, it is not known to what extent the plasmid DNA catabolism is subordinated to the catabolism of the vector. In the work reported here, the problem was approached by following the intracellular fate in rat liver, of plasmid [35S]DNA complexed with a cationic peptide poly-L-lysine that can be hydrolyzed by cellular peptidases or with its stereoisomer, poly-D-lysine, that cannot be split by these enzymes. Complexes of DNA with poly-L-lysine and poly-D-lysine are taken up to the same extent by the liver, mainly by Kupffer cells, but the intracellular degradation of nucleic acid molecules is markedly quicker when poly-L-lysine is injected. The association of DNA with the polycations inhibits DNA hydrolysis in vitro by purified lysosomes but similarly for poly-L-lysine and poly-D-lysine. The intracellular journey followed by [35S]DNA complexed with poly-L- or poly-D-lysine was investigated using differential and isopycnic centrifugation. Results indicate that [35S]DNA is transferred more slowly to lysosomes, the main site of intracellular degradation of endocytosed macromolecules, when it is given as a complex with poly-D-lysine than with poly-L-lysine. They suggest that the digestion of the vector in a prelysosomal compartment is required to allow endocytosed plasmid DNA to rapidly reach lysosomes. Such a phenomenon could explain why injected plasmid DNA is more stable in vivo when it is associated with poly-D-lysine.

We have studied the localization of mutant cystic fibrosis transmembrane regulator delta F508CFTR in pancreatic adenocarcinoma cells (CFPAC), which naturally express the mutant protein. Our goal was to investigate whether delta F508CFTR is strictly retained in the endoplasmic reticulum (ER) or alternatively whether it can be transported beyond the ER and reach the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). This compartment, defined by the presence of the 53-kDa protein ERGIC-53, was identified by subcellular fractionation and by immunofluorescence. Part of the delta F508CFTR population and ERGIC-53 showed similar distributions in membrane fractions analyzed on Nycodenz density gradients. Both proteins were present in density fractions distinct from the ones containing the ER marker proteins calnexin and Sec61. Immunofluorescence microscopy of CFPAC cells revealed some colocalization of delta F508CFTR with ERGIC-53. Following incubation of CFPAC cells at 15 degrees C, a condition known to block ER to Golgi transport, both ERGIC-53 and delta F508CFTR subcellular localizations were altered. By contrast, this temperature shift had no effect on the localization of the ER marker Sec61. Our observations indicate that the abnormal protein delta F508CFTR can leak out of the ER and reach the ERGIC. These results support the idea that this intermediate compartment plays a role in the trafficking events leading to retention and finally degradation of the misfolded delta F508CFTR protein.

Lysosomal-membrane-associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell-surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E-selectin and between Lamps and galectins (endogenous-galactoside-binding lectins). The present study examined the expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell-surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo-2 (human colon-adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin-3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin-3 binding to Lamps-expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis.

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N-(1-(2,3-dioleoxy)propyl)-N,N,N,-trimethylammonium methyl-sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of beta-galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.

Most lysosomal hydrolases are soluble enzymes. Lamp-II (lysosome-associated membrane protein-II) is a major constituent of the lysosomal membrane. We studied the aggregation of a series of lysosomal molecules. The aggregation-sensitive lysosomal marker enzymes were optimally aggregated at intralysosomal pH. A similar pH dependence was recorded for aggregation of Lamp-II. The pH-dependent loss of solubility of isolated Lamp-II required components of the lysosome extract. Conditions of mild acid pH promoting aggregation triggered the formation of complexes with lipids of lysosomal origin. We fractionated a membrane-free lysosome extract by gel-filtration chromatography and could reconstitute assemblies in vitro from separated fractions. We found some selectivity in the lysosomal proteins binding to complex lipids, phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine being most effective. We propose that the formation at pH 5.0 of such supramolecular assemblies between lysosomal proteins and lipids occurs within the intralysosomal environment. Some possible consequences of such an intralysosomal matrix formation on organelle function are discussed.

Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

Lamp II (for lysosomal associated membrane protein II) is an integral type I glycoprotein. It consists of a very large and heavily glycosylated luminal domain, a single transmembrane segment, and a short cytoplasmic tail. We show that in highly purified lysosomes from rat liver, Lamp II, immunodetected with a monoclonal antibody on Western blots, it surprisingly distributed between a membrane bound form and a "soluble" form. The partition of the protein between the membrane and the content of lysosomes is strongly pH dependent. The soluble Lamp II population is sensitive to pH dependent aggregation as it is for many lysosomal content enzymes.

To study the transfer of phagocytosed components from phagosomes to lysosomes, we have investigated phagocytosis by rat liver of killed Staphylococcus aureus labelled with (125)I tyramine cellobiose. Lysosomes were identified by injecting the animals with Triton WR1339, a non ionic detergent that is endocytosed by the liver and accumulates in lysosomes, causing a marked decrease of their density; that allows these organelles to be well separated from other particles in a density gradient. Bacteria were quickly taken up by the liver; their uptake is followed by a slow degradation as ascertained by the increase of acid-soluble radioactivity in the homogenates with time. Triton WR1339 injection does not affect the uptake and the degradation of the particles. Differential centrifugation of homogenates shows that at any time after injection, most of the radioactivity is recovered in the mitochondrial fractions. Distributions of acid precipitable and acid soluble radioactivities amongst subcellular structures present in mitochondrial fractions were studied by isopycnic centrifugation in sucrose gradients, at increasing times after bacteria injection. Results show that: 1) acid-precipitable radioactivity is quasi-exclusively present in gradient fractions of high density, well separated from the fractions where there are recovered lysosomes; 2) with time, acid-soluble radioactivity is more and more associated with lysosomes, however, a significant proportion can be detected for many hours after injection, in gradient fractions where acid-precipitable radioactivity is located. The most plausible explanation of our observations is that phagocytosed particles are degraded in phagosomes and that the degradation products are delivered to lysosomes, probably by a vesicular process.

Previous studies have demonstrated that reactive oxygen species are involved in ischemic injury. The present work was undertaken to determine in vivo the role of xanthine oxidase in the oxygen free radical production during rat liver ischemia and to examine the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) during the same period. Our results indicate a 4-fold increase in xanthine oxidase activity between 2 and 3 hours of normothermic ischemia, in parallel with a decrease in cell viability. Moderate hypothermia delays both events. Under the same conditions, the activity of oxygen radical scavenging enzymes remains unchanged. Moreover, we have compared in vitro the susceptibility of isolated liver cells to an oxidative stress induced by O2.-, H2O2 and .OH. Our results reveal that endothelial cells are much more susceptible to reactive oxygen species than hepatocytes, probably because they lack H2O2-detoxifying enzymes. These findings suggest that xanthine oxidase might play a major role in the ischemic injury mainly at the level of the sinusoidal space where most endothelial cells are located.

Both freshly-isolated rat hepatocytes and Morris hepatoma 7777 cells synthesized cathepsin D as a precursor that was either processed intracellular to smaller mature forms or secreted into the medium. The pattern of mature enzyme forms was different in the 2 cell types. In addition, the relative amount of precursor secreted was much higher for hepatoma cells. Monensin strongly enhanced the secretion and also impaired the intracellular transport-linked maturation of procathepsin D in hepatocytes, while it markedly inhibited intracellular maturation and only slightly increased secretion of the pro-enzyme in hepatoma cells. Ammonium chloride influenced the intralysosomal segregation and maturation of procathepsin D in hepatocytes but not in hepatoma cells. Our observations indicate that (i) the lysosomal segregation of cathepsin D was less efficient and its fractional secretion higher in hepatoma cells than in hepatocytes; (ii) in the 2 cell types, delivery to lysosomes and processing of procathepsin D were differently sensitive to increases in the vacuolar pH.

The present results showed that uptake of dopamine (DA) by rat isolated hepatocytes was mediated, in addition to simple diffusion, mainly by a transporter-involved process, with Km of 66.8 mumol and Vmax of 52.3 pmol.min-1/10(5) cells. The process was pH- and temperature-dependent and required an activation energy of 4.12 kcal.mol-1 (Q10 = 1.25) in the range of 2.0-12.7 C and 13.0 kcal.mol-1 (Q10 = 2.0) in the range of 12.7-39.0 C. Cysteine residue having free thiol group was unrelated to the activity of the transporter. Catecholamines, serotonin, and cocaine inhibited the DA transport, but tyramine (TA) and tryptamine, as well as benztropine and imipramine (which are potent inhibitors for hepatic TA transporter and neuronal DA transporter), had no inhibitory effect on the transport of DA in these cells. These results indicated that DA was taken up into hepatocytes by a distinct carrier. NaF and mastoparan influenced the transport activity in these cells further, suggesting that signal transducing G-proteins may be involved in the regulation of DA transporter in rat hepatocytes.

In the work reported here, we have compared the elimination from the blood, the uptake by the liver and the intracellular distribution of bovine growth hormone, free(Gh) or bound to a monoclonal antibody (GhAb). Results show that: a) the elimination from the blood is more rapid for Gh than for GhAb; b) both molecules are quickly taken up by the liver; c) probably after travelling through endosomes, Gh and GhAb get to lysosomes where they are degraded. However, Gh mostly ends in hepatocyte lysosomes while GhAb is recovered to a large extent in sinusoidal cell lysosomes; and d) binding by isolated hepatocytes is markedly less efficient for GhAb than for Gh.

Observations on the uptake of tyramine by hepatocytes indicate that the amine is taken up by simple diffusion and a transporter mediated system, with a Km of 39 microM and a Vmax of 270 pmol/min/10(5) cells. The carrier-mediated process is pH- and temperature-dependent and requires an activation energy of 12.9 kcal/mol. An overshoot uptake is achieved a few minutes after adding this amine to the cell suspension, suggesting that active transport is involved. This is supported by the finding that partial inhibition of the uptake can be induced by oligomycin, azide, cyanide and dinitrophenol. NO3-, SCN- and SO4(2-), which change the membrane potential significantly, and depress the transporter mediated uptake further, suggesting that the membrane potential is the driving force for the entry of this amine across hepatic membrane. Cysteine is essential for the normal carrier function; whereas, histidine, tryptophan, arginine and lysine do not directly deal with the activity of the carrier. Many substances, but not amino acids, H, M, and N receptor agonists, can inhibit the uptake of tyramine. It is possible that other amines can enter hepatocytes by using this transporter.

When a light mitochondrial fraction (L fraction) of rat liver is incubated in the presence of an oxygen free radical generating system (xanthine-xanthine oxidase), the free activity of N-acetylglucosaminidase (NAGase) increases as a result of the deterioration of the lysosomal membrane. Various flavonoids are able to prevent this phenomenon, others are ineffective. Comparative activity studies suggest the importance of the presence of two OH groups in orthosubstitution in the B ring and of an OH in the 3 position. Flavan-type flavonoids behave like their related flavonoids; d-catechin also opposes lysosome disruption. Kaempferol, quercetin, 7,8-dihydroxyflavone and d-catechin inhibit lipoperoxidation occurring in an L fraction incubated with the xanthine oxidase system as ascertained by malondialdehyde (MDA) production. For kaempferol and quercetin, such an inhibition parallels the prevention of NAGase release; this is not the case for the two other compounds where inhibition of NAGase release takes place at a flavonoid concentration lower than that required to oppose MDA production. Morphological observations performed on purified lysosomes confirm the biochemical results. Some flavonoids are also able to prevent release of NAGase caused by the incubation of an L fraction in isoosmotic glucose. Only flavone and hydroxyflavones are effective. It is proposed that the protective effect of flavonoids on lysosomes subjected to oxygen free radicals does not only originate from their scavenger and antilipoperoxidant properties; a more direct action on lysosomal membrane making it more resistant to oxidative aggression has to be considered. The prevention by some flavonoids of lysosome osmotic disruption in isoosmotic glucose could be the result of an inhibition of glucose translocation through the lysosomal membrane.

The uptake of 125I-tyramine cellobiose (TC) by isolated rat hepatocytes and by total rat liver is markedly higher than that of 14C-sucrose and 125I-PVP, suggesting that TC does not enter the cells by fluid phase endocytosis. The distribution of radioactivity after differential centrifugation shows that the compound is shared out amongst sedimentable structures and unsedimentable fraction. Analysis by isopycnic centrifugation indicates that quickly after its penetration into the cells, most of sedimentable 125I-TC is associated with lysosomes. Such an intracellular localization is confirmed by the distributions observed after free flow electrophoresis and by the fact that radioactivity and cathepsin C, a lysosomal hydrolase, are simultaneously released from a mitochondrial fraction treated with glycyl-L-phenylalanine-2-naphthylamide. Pretreatment of the rats with chloroquine, an acidotropic drug that accumulates in lysosomes, prevents to some extent the entry of 125I-TC into these organelles. Experiments performed with purified lysosomes show that 14C-sucrose does not cross the lysosomal membrane when 125I-TC accumulates linearly with time in the fractions. These results are explained by supposing that the linkage of tyramine to cellobiose allow the disaccharide to diffuse through the plasma and the lysosome membranes, and that the accumulation of the molecule in these organelles results from its weak basic properties. 125I-TC could be an interesting molecule with which to study acidotropism in the whole animal and in isolated and cultured cells.

We have investigated the endocytosis by rat liver of superoxide dismutase (SOD) labelled with 125I. (125I) SOD is quickly taken up by the liver where it remains in significant amounts for at least 150 min. Adsorptive endocytosis is probably involved. Distribution of radioactivity was established after differential and isopycnic centrifugation and compared with that of cathepsin C, a lysosomal enzyme. Results show that the behavior of radioactivity is similar to that of the hydrolase. SOD activity is only marginally affected by incubation in the presence of a purified lysosome extract; moreover, when (125I) SOD is treated in the same conditions, only a few percent of radioactivity becomes acidosoluble. These observations indicate that SOD taken up by the liver accumulates in lysosomes where it can stay for a relatively long time owing to its relative resistance to lysosomal hydrolases.

By a variety of methods, such as ultracentrifugation in different media, hypoosmotic activation and hydrostatic compression, subcellular particles were characterized at different perinatal ages and compared to the adult rats. Fetal mitochondria elicited a higher density, an increased osmotic space and a greater resistance to compression. The size of these particles was larger than in the adults. Mitochondria in the 1-day-old animals were freely permeable to sucrose and their external membrane was more resistant to hypoosmotic activation. Lysosomes were shown to decrease their sucrose permeability and their resistance to hypoosmotic activation with development. Moreover, the size of the lysosomes increased with development.

The influence of culture duration on some characteristics of hepatocyte subcellular membranes involved in endocytosis was investigated. Activity of enzymes located in plasma membrane, Golgi apparatus and lysosomes increases with time. These modifications are accompanied with several changes in the sedimentation properties of these organelles. Endocytosis of [14C]sucrose and [14C]sucrose-LDL is not affected by culture age. On the contrary, [14C]sucrose-ASF endocytosis strongly decreases in these conditions. These modifications are delayed to some extent by lowering the temperature. Addition to the culture medium of 3-methyladenine (an inhibitor of autophagy), sodium butyrate, dimethylsulfoxide, phenobarbital or nicotinamide does not prevent the decrease of ASF endocytosis caused by culture duration. These results indicate that one must be cautious when extrapolating to liver in vivo, observations on endocytosis obtained with primary culture of hepatocytes.

Plant proteases are essential enzymes that play key roles during crucial phases of plant life. Some proteases are mainly involved in general protein turnover and recycle amino acids for protein synthesis. Other proteases are involved in cell signalling, cleave specific substrates and are key players during important genetically controlled molecular processes. Cathepsin B is a cysteine protease that can do both because of its exopeptidase and endopeptidase activities. Animal cathepsin B has been investigated for many years, and much is known about its mode of action and substrate preferences, but much remains to be discovered about this potent protease in plants. Cathepsin B is involved in plant development, germination, senescence, microspore embryogenesis, pathogen defence and responses to abiotic stress, including programmed cell death. This review discusses the structural features, the activity of the enzyme and the differences between the plant and animal forms. We discuss its maturation and subcellular localisation and provide a detailed overview of the involvement of cathepsin B in important plant life processes. A greater understanding of the cell signalling processes involving cathepsin B is needed for applied discoveries in plant biotechnology. [ABSTRACT FROM AUTHOR]

Approximately 75 % of marketed drugs have the physicochemical property of being weak bases. Weak-base drugs with relatively high pKa values enter acidic organelles including endosomes and lysosomes (endolysosomes), reside in and de-acidify endolysosomes, and induce cytotoxicity. Divalent cations within endolysosomes, including iron, are released upon endolysosome de-acidification. Endolysosomes are "master regulators of iron homeostasis", and neurodegeneration is linked to ferrous iron (Fe2+)-induced reactive oxygen species (ROS) generation via Fenton chemistry. Because endolysosome de-acidification-induced lysosome-stress responses release endolysosome Fe2+, it was crucial to determine the mechanisms by which a functionally and structurally diverse group of weak base drugs including atropine, azithromycin, fluoxetine, metoprolol, and tamoxifen influence endolysosomes and cause cell death. Using U87MG astrocytoma and SH-SY5Y neuroblastoma cells, we conducted concentration-response relationships for 5 weak-base drugs to determine EC50 values. From these curves, we chose pharmacologically and therapeutically relevant concentrations to determine if weak-base drugs induced lysosome-stress responses by de-acidifying endolysosomes, releasing endolysosome Fe2+ in sufficient levels to increase cytosolic and mitochondria Fe2+ and ROS levels and cell death. Atropine (anticholinergic), azithromycin (antibiotic), fluoxetine (antidepressant), metoprolol (beta-adrenergic), and tamoxifen (anti-estrogen) at pharmacologically and therapeutically relevant concentrations (1) de-acidified endolysosomes, (2) decreased Fe2+ levels in endolysosomes, (3) increased Fe2+ and ROS levels in cytosol and mitochondria, (4) induced mitochondrial membrane potential depolarization, and (5) increased cell death; effects prevented by the endocytosed iron-chelator deferoxamine. Weak-base pharmaceuticals induce lysosome-stress responses that may affect their safety profiles; a better understanding of weak-base drugs on Fe2+ interorganellar signaling may improve pharmacotherapeutics. [ABSTRACT FROM AUTHOR]

Although it has been known for decades that lysosomes are central for degradation and recycling in the cell, their pivotal role as nutrient sensing signaling hubs has recently become of central interest. Since lysosomes are highly dynamic and in constant change regarding content and intracellular position, fusion/fission events allow communication between organelles in the cell, as well as cell-to-cell communication via exocytosis of lysosomal content and release of extracellular vesicles. Lysosomes also mediate different forms of regulated cell death by permeabilization of the lysosomal membrane and release of their content to the cytosol. In cancer cells, lysosomal biogenesis and autophagy are increased to support the increased metabolism and allow growth even under nutrient- and oxygen-poor conditions. Tumor cells also induce exocytosis of lysosomal content to the extracellular space to promote invasion and metastasis. However, due to the enhanced lysosomal function, cancer cells are often more susceptible to lysosomal membrane permeabilization, providing an alternative strategy to induce cell death. This review summarizes the current knowledge of cancer-associated alterations in lysosomal structure and function and illustrates how lysosomal exocytosis and release of extracellular vesicles affect disease progression. We focus on functional differences depending on lysosomal localization and the regulation of intracellular transport, and lastly provide insight how new therapeutic strategies can exploit the power of the lysosome and improve cancer treatment. [ABSTRACT FROM AUTHOR]

The immune system acts as a defense mechanism against foreign antigens. Impairment of the immune system leads to the development of chronic diseases such as respiratory infections, cancer, cardiovascular diseases, and neurodegeneration. Macrophages, natural scavengers that are part of innate immunity, are known to directly participate in scavenging foreign antigens. The functional modulation of macrophages could be an effective treatment for pathogens. Seaweeds are marine macroalgae known to exhibit multiple bioactive properties. Thus, this study evaluated the immune-enhancing properties of marine brown algae extracts of Sargassum horneri (SH), Undaria pinnatifida (UP), and Sargassum fusiforme (SF) on murine macrophage cells. The results showed that all three algal extracts stimulated cell proliferation. SH and UP outshined SF in enhancing the expression levels of IL-1β, TNF-α, and IL-6 at almost all the concentrations tested as compared to SF which showed similar effects only at 200 or 400 μg/mL. A similar trend was seen in TNF-α, NO, and PGE2 production. Additionally, only SH and SF could enhance the mRNA expression levels of IL-12, and only SH upregulated the mRNA expression level of IL-10. The algal extracts also enhanced the phagocytosis activity of macrophages at 50–400 μg/mL for SH and 100–400 μg/mL for UP and SF. In conclusion, we found that these algal extracts could be considered immunomodulators that enhance the functional activity of macrophages. [ABSTRACT FROM AUTHOR]

Autosomal recessive cerebellar ataxias (ARCAs) are a heterogeneous group of neurodegenerative disorders affecting primarily the cerebellum and/or its afferent tracts, often accompanied by damage of other neurological or extra-neurological systems. Due to the overlap of clinical presentation among ARCAs and the variety of hereditary, acquired, and reversible etiologies that can determine cerebellar dysfunction, the differential diagnosis is challenging, but also urgent considering the ongoing development of promising target therapies. The examination of afferent and efferent visual system may provide neurophysiological and structural information related to cerebellar dysfunction and neurodegeneration thus allowing a possible diagnostic classification approach according to ocular features. While optic coherence tomography (OCT) is applied for the parametrization of the optic nerve and macular area, the eye movements analysis relies on a wide range of eye-tracker devices and the application of machine-learning techniques. We discuss the results of clinical and eye-tracking oculomotor examination, the OCT findings and some advancing of computer science in ARCAs thus providing evidence sustaining the identification of robust eye parameters as possible markers of ARCAs. [ABSTRACT FROM AUTHOR]

Niemann–Pick Disease (NPD) is a rare autosomal recessive disease belonging to lysosomal storage disorders. Three types of NPD have been described: NPD type A, B, and C. NPD type A and B are caused by mutations in the gene SMPD1 coding for sphingomyelin phosphodiesterase 1, with a consequent lack of acid sphingomyelinase activity. These diseases have been thus classified as acid sphingomyelinase deficiencies (ASMDs). NPD type C is a neurologic disorder due to mutations in the genes NPC1 or NPC2, causing a defect of cholesterol trafficking and esterification. Although all three types of NPD can manifest with pulmonary involvement, lung disease occurs more frequently in NPD type B, typically with interstitial lung disease, recurrent pulmonary infections, and respiratory failure. In this sense, bronchoscopy with broncho-alveolar lavage or biopsy together with high-resolution computed tomography are fundamental diagnostic tools. Although several efforts have been made to find an effective therapy for NPD, to date, only limited therapeutic options are available. Enzyme replacement therapy with Olipudase α is the first and only approved disease-modifying therapy for patients with ASMD. A lung transplant and hematopoietic stem cell transplantation are also described for ASMD in the literature. The only approved disease-modifying therapy in NPD type C is miglustat, a substrate-reduction treatment. The aim of this review was to delineate a state of the art on the genetic basis and lung involvement in NPD, focusing on clinical manifestations, radiologic and histopathologic characteristics of the disease, and available therapeutic options, with a gaze on future therapeutic strategies. [ABSTRACT FROM AUTHOR]

Simple Summary: This article focuses on the impairment of lysosomes in the context of lysosomal storage disorders. Lysosomal storage disorders are a group of rare diseases with different causes united by the malfunctioning of the lysosomes. Lysosomes are intracellular vesicles, and their main function is to decompose intracellular waste in a process known as autophagy. Besides this function, lysosomes participate in a wide range of essential mechanisms aimed to keep the internal balance within our cells, which is called homeostasis. Maintaining homeostasis is a fundamental goal of all biological systems, from the simplest to the most complex, and is essential for proper functioning. Many of these disorders are originated from single-gene mutations. This provides a valuable starting point for scientists to trace the path from a single molecule to disease symptoms across the complexity of living organisms. The purpose of this paper is to provide insights from the molecular to the clinical level on this group of diseases, focusing on changes in autophagy and the latest therapeutic approaches in the field. Lysosomes are the main organelles responsible for the degradation of macromolecules in eukaryotic cells. Beyond their fundamental role in degradation, lysosomes are involved in different physiological processes such as autophagy, nutrient sensing, and intracellular signaling. In some circumstances, lysosomal abnormalities underlie several human pathologies with different etiologies known as known as lysosomal storage disorders (LSDs). These disorders can result from deficiencies in primary lysosomal enzymes, dysfunction of lysosomal enzyme activators, alterations in modifiers that impact lysosomal function, or changes in membrane-associated proteins, among other factors. The clinical phenotype observed in affected patients hinges on the type and location of the accumulating substrate, influenced by genetic mutations and residual enzyme activity. In this context, the scientific community is dedicated to exploring potential therapeutic approaches, striving not only to extend lifespan but also to enhance the overall quality of life for individuals afflicted with LSDs. This review provides insights into lysosomal dysfunction from a molecular perspective, particularly in the context of human diseases, and highlights recent advancements and breakthroughs in this field. [ABSTRACT FROM AUTHOR]

Glycyl-D-phenylalanine-2-naphthylamide (Gly-D-Phe-2-NNap) is a cytotoxic agent as exemplified by its effect on Vero cells in culture. This effect is inhibited to some extent by nigericin. On the other hand, Gly-D-Phe-2-NNap induces an increase of free activity of N-acetylglucosaminidase when incubated with a mitochondrial fraction of rat liver at pH 7.5. The phenomenon is inhibited by chloroquine, NH4Cl and nigericin, substances that are known to increase the intralysosomal pH. The latency of enzymes located in other subcellular structures - mitochondria, peroxisomes and endoplasmic reticulum - is not affected by Gly-D-Phe-2-NNap. Moreover, that compound does not cause a release of FITC-Dextran present in endosomes. Apparently Gly-D-Phe-2-NNap is a specific lytic agent for lysosomes. It is proposed that the molecule behaves like a lysosomotropic substance that is able to attack the lysosomal membrane from the interior of the organelle. Its cytotoxic properties could be explained by its effect on lysosomes.