Acarbose is a competitive inhibitor of mammalian lysosomal acid alpha-D-glucosidases

Intraperitoneal injections (approximately 400 mg/kg of body weight) of acarbose, an inhibitor of acid (1----4)-alpha-D-glucosidase, perturb the metabolism of glycogen in the liver, resulting in excess storage of lysosomal glycogen. The metabolism of skeletal muscle glycogen was unaffected, suggesting that acarbose either does not enter the tissue or that the muscle alpha-D-glucosidase is not inhibited. The hydrolysis of maltose and glycogen by the acid alpha-D-glucosidases from rat liver, rat skeletal muscle, and human placenta was inhibited competitively by acarbose. Thus, the lack of effect of acarbose upon the metabolism of muscle glycogen is due to its inability to enter the tissue.     

Centripetal cholesterol flow from the extrahepatic organs through the liver is normal in mice with mutated Niemann-Pick type C protein (NPC1)

Niemann-Pick type C (NPC) protein functions to move unesterified cholesterol from the lysosomal compartment to other intracellular sites for further metabolism and/or excretion. This cholesterol is brought into the cell through the coated-pit pathway and accumulates in the lysosomes when NPC protein is mutated. The present study quantitated the alternative uptake process that brings cholesterol into the cell through the scavenger receptor, class B, type I (SR-BI) pathway in animals with this mutation. In homozygous NPC mice, the tissues of the extrahepatic compartment accumulated an excess of 14 mg of cholesterol each day per kg body weight, and synthesis increased by a similar amount (to 111 mg/day per kg) to compensate for this functional loss of sterol through lysosomal sequestration. An amount of cholesterol (108 mg/day per kg) nearly equal to that synthesized in the extrahepatic compartment was carried through the circulation by high density lipoprotein (HDL) and taken up by the liver. The rate of hepatic cholesterol excretion from the NPC mice as fecal acidic (65 mg/day per kg) and neutral (85 mg/day per kg) sterols was elevated 61% above control values and was accounted for by the total amount of cholesterol brought to the liver in HDL and synthesized in the hepatocytes. These studies demonstrated that while cholesterol entering tissues of the NPC animals through the coated-pit pathway became sequestered in the lysosomal compartment and was metabolically inactive, cholesterol that was newly synthesized or that entered cells through the SR-BI pathway was metabolized and excreted normally.     

The values and limits of an in vitro model of Pompe disease: The best laid schemes o’ mice an’ men ……..†

In Pompe disease, a lysosomal glycogen storage disorder, cardiac and skeletal muscle abnormalities are responsible for premature death and severe weakness. Swollen glycogen-filled lysosomes, the expected pathology, are accompanied in skeletal muscle by a secondary pathology – massive accumulation of autophagic debris – that appears to contribute greatly to the weakness. We have tried to reproduce these defects in murine, Pompe myotubes derived from either primary myoblasts or myoblasts with extended proliferative capacity. The cells accumulated large lysosomes filled with glycogen, but, to our disappointment, did not have autophagic buildup even though basal autophagy was intact. When we suppressed autophagy by knocking down Atg7, we found that glycogen uptake by lysosomes was not affected, suggesting that macroautophagy is not the major pathway for glycogen delivery to lysosomes. But two apparently incidental observations – a peculiar distribution of both microinjected dextran and of small acidic structures adjacent to the interior membrane of large alkalinized glycogen-containing lysosomes – raised the possibility that glycogen traffics to the lysosomes by microautophagy or/and by the engulfment of small lysosomes by large ones. The cultured myotubes, therefore, appear to be a useful model for studying the mechanisms involved in glycogen accumulation in Pompe disease and to test substrate deprivation approaches.

^?From To a Mouse, Robert Burns, 1785     

急性高原性低氧对三种小哺乳动物肝脏作用的比较

人工低气压舱模拟高原低氧,观察实验动物小白鼠、豚鼠及野生动物达乌尔鼠兔,在海拔5000米及8000米24小时内的几种生理效应,并与2300米海拔对照组进行了比较。发现随着海拔高度的升高:1.3种动物体重明显下降;2.肝糖原含量明显降低:小鼠、达乌尔鼠兔与豚鼠肝糖原在8000米时分别为2300米含量的62%,35%及 9%:3.小鼠、达乌尔鼠兔及豚鼠的肝脂肪累积量依次增大;4.肝蛋白质含量减少;5.SGPT与SGOT活力升高;6.肝细胞溶酶体的酸性磷酸酶与芳基硫酸脂酶活力升高。肝组织形态学观察结果与生化检测结果一致,豚鼠在8000米海拔时,肝细胞出现气球样变,脂肪变性及灶性液化性坏死等变化。综合分析,这3种动物对极度低氧的耐受性依顺序为小鼠、达乌尔鼠兔、豚鼠。     

The existence of a lysosomal redox chain and the role of ubiquinone

Several studies concerning the distribution of ubiquinone (UQ) in the cell report a preferential accumulation of this biogenic quinone in mitochondria, plasma membranes, Golgi vesicles, and lysosomes. Except for mitochondria, no recent comprehensive experimental evidence exists on the particular function of UQ in these subcellular organelles. The aim of a recent study was to elucidate whether UQ is an active part of an electron-transfer system in lysosomes. In the present work, a lysosomal fraction was prepared from a light mitochondrial fraction of rat liver by isopycnic centrifugation. The purity of our preparation was verified by estimation of the respective marker enzymes. Analysis of lysosomes for putative redox carriers and redox processes in lysosomes was carried out by optical spectroscopy, HPLC, oxymetry, and ESR techniques. UQ was detected in an amount of 2.2 nmol/mg of protein in lysosomes. Furthermore, a b-type cytochrome and a flavin-adenine dinucleotide (FAD) were identified as other potential electron carriers. Since NADH was reported to serve as a substrate of UQ redox chains in plasma membranes, we also tested this reductant in lysosomes. Our experiments demonstrate a NADH-dependent reduction of UQ by two subsequent one-electron-transfer steps giving rise to the presence of ubisemiquinone and an increase of the ubiquinol pool in lysosomes. Lysosomal NADH oxidation was accompanied by an approximately equimolar oxygen consumption, suggesting that O(2) acts as a terminal acceptor of this redox chain. DMPO/(*)OH spin adducts were detected by ESR in NADH-supplemented lysosomes, suggesting a univalent reduction of oxygen. The kinetic analysis of redox changes in lysosomes revealed that electron carriers operate in the sequence NADH > FAD > cytochrome b > ubiquinone > oxygen. By using the basic spin label TEMPAMINE, we showed that the NADH-related redox chain in lysosomes supports proton accumulation in lysosomes. In contrast to the hypothesis that UQ in lysosomes is simply a waste product of autophagy in the cell, we demonstrated that this lipophilic electron carrier is a native constituent of a lysosomal electron transport chain, which promotes proton translocation across the lysosomal membrane.     

Post mortem glycogenolysis is a combination of phosphorolysis and hydrolysis

1. Glycogen, glucose, lactate and glycogen phosphorylase concentrations and the activities of glycogen phosphorylase a and acid 1,4-alpha-glucosidase were measured at various times up to 120 min after death in the liver and skeletal muscle of Wistar and gsd/gsd (phosphorylase b kinase deficient) rats and Wistar rats treated with the acid alpha-glucosidase inhibitor acarbose. 2. In all tissues glycogen was degraded rapidly and was accompanied by an increase in tissue glucose and lactate concentrations and a lowering of tissue pH. In the liver of Wistar and acarbose-treated Wistar rats and in the skeletal muscle of all rats glycogen loss proceeded initially very rapidly before slowing. In the gsd/gsd rat liver glycogenolysis proceeded at a linear rate throughout the incubation period. Over 120 min 60, 20 and 50% of the hepatic glycogen store was degraded in the livers of Wistar, gsd/gsd and acarbose-treated Wistar rats, respectively. All 3 types of rat degraded skeletal muscle glycogen at the same rate and to the same extent (82% degraded over 2 hr). 3. In Wistar rat liver and skeletal muscle glycogen phosphorylase was activated soon after death and the activity of phosphorylase a remained well above the zero-time level at all later time points, even when the rate of glycogenolysis had slowed significantly. Liver and skeletal muscle acid alpha-glucosidase activities were unchanged after death. 4. The decreased rate and extent of hepatic glycogenolysis in both the gsd/gsd and acarbose-treated rats suggests that this process is a combination of phosphorolysis and hydrolysis. 5. Glycogen was purified from Wistar liver and skeletal muscle at various times post mortem and its structure investigated. Fine structural analysis revealed progressive shortening of the outer chains of the glycogen from both tissues, indicative of random, lysosomal hydrolysis. Analysis of molecular weight distributions showed inhomogeneity in the glycogen loss; in both tissues high molecular weight glycogen was preferentially degraded. This material is concentrated in lysosomes of both skeletal muscle and liver. These results are consistent with a role for lysosomal hydrolysis in glycogen degradation.     

Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation.

1. A crude lysosomal fraction obtained by differential centrifugation of a rat liver homogenate was subjected to zonal centrifugation in iso-osmotic self-generating gradients composed of modified colloidal silica (Percoll). Analysis of relevant marker-enzyme activities shows a continuous band of considerably purified lysosomal particles in the density range 1.04--1.11 g/ml. 2. A relationship between age and buoyant density of the parenchymal lysosomal subpopulations is indicated by the distribution of 125I-labelled asialoglycoproteins in the heterogeneous lysosomes during the catabolism of the glycoprotein. The labelled asialoglycoprotein first appeared in lysosomal particles of low density, which with time progressively acquired a higher density. Furthermore, 30 min after administration the 125I-labelled asialocaeruloplasmin recovered in the light lysosomes was less degraded than the material recovered in the heavy lysosomes. 3. A lysosomal enzyme (arylsulphatase) was found to possess considerably higher isoelectric points in the heavy lysosomes than in the light lysosomes, which is consistent with a relationship between age and density of the lysosomes.     

Changes in lysosome populations in the rat kidney cortex induced by experimental proteinuria

1. Experimental proteinuria (262.9 mg protein/24 hr urine) was induced in rats by repeated intraperitoneal injections of BSA. 2. Hypertrophy of the kidney cortex was significant 8 days after the start of the BSA injections, and the activities of lysosomal enzymes in kidney cortex and urine were significantly higher in proteinuric compared to nonproteinuric rats. 3. Lysosome populations in the kidney cortex were examined by rate sedimentation of the homogenate and by rate zonal and isopycnic centrifugation of the lysosome-rich ML fraction. 4. The activity of lysosomal enzymes in the kidney cortex increased slightly, essentially in the large, fragile lysosomes mainly recovered from the proximal tubule. 5. Proteinuria induced a shift/reduction in the density of small lysosomes from 1.235 and 1.20 g/ml to 1.225 and 1.185 g/ml, respectively. 6. Proteinuria induced a new population of small lysosomes (density 1.185 g/ml) enriched in cathepsin D.     

Glycogen metabolism in the liver of the developing rat.

1. The total activity of glycogen synthease increased 20-fold from day 17 of gestation to birth at day 22, with a further increase of 18% in the 24h after birth. Active synthase (I) rose 45-fold to a maximum at day 21, fell 40% before birth, and then increased by a similar amount 24h after birth. The fraction of synthase in the active form correlated very well with the deposition of glycogen in the liver. 2. Total phosphorylase had a similar developmental pattern of total synthease with an 18-fold increase from day 17 to day 22. The appearance of active phosphorylase showed a lag-period compared with total phosphorylase and did not increase significantly until day 20. The fraction of phosphorylase in the active form did not correlate at all with glycogen deposition or mobilization. 3. There was a close relationshp between the ratio of phosphorylase a/synthase I and the glycogen content of the liver. An increase or decrease in this ratio would result in glycogenolysis of glycogenesis respectively. 4. It is postulated that a cycle between the two enzymes under basal conditions could exist which permits a continuous turnover of glycogen. Such a system would explain why active phosphorylase is always seen, even under conditions of net glycogen synthesis. The differences in hormone sensitivity of synthase and phosphorylase would also be accounted for as only one enzyme would have to respond acutely to hormonal influences.     

Lysosomal cholesterol accumulation in macrophages leading to coronary atherosclerosis in CD38−/− mice

The disruption in transportation of oxLDL‐derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca²⁺ messenger of nicotinic acid adenine dinucleotide phosphate (NAADP), an enzymatic product of CD38 ADP‐ribosylcyclase (CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that oxLDL concentration‐dependently increased lipid buildup in bone marrow‐derived macrophages from both wild type and CD38^?/?, but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38^?/? mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38^?/? mice.     

Regression of autophagic vacuoles in pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells: a comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment

Treatment of mice with both leupeptin (0.06 mg/g body wt) and vinblastine (0.05 mg/g body wt) for 2 h caused a many-fold enlargement of the autophagic-lysosomal compartment of pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells. In all three types of cells a predominance of large, dense bodies was seen after leupeptin treatment and that of typical autophagic vacuoles were seen after vinblastine treatment. An exponential decrease of the volume fraction of autophagic vacuoles was observed in leupeptin-treated cells after the administration of cycloheximide (0.2 mg/g body wt). The half-life of autophagic vacuoles estimated from the decay curve was 5.3, 5.7, and 6.6 min for pancreatic, seminal vesicle, and liver cells, respectively. Our data suggest that sequestered cytoplasmic material rapidly enters the lysosomes in leupeptin-treated cells and accumulates in this compartment. In contrast, no regression of the autophagic vacuole compartment of pancreatic and seminal vesicle cells was observed after the administration of cycloheximide to animals pretreated with vinblastine, and only a slight decrease was seen in liver cells. These observations show that the lifetime of autophagic vacuoles is prolonged by vinblastine resulting in their accumulation in the cells. However, our measurements also lend support to the view that in addition to the accumulatory effect on undegraded cytoplasmic material, stimulation of sequestration may play a role in the enlargement of the autophagic lysosomal compartment after treatment with leupeptin as well as with vinblastine in all three types of cells investigated.     

Studies of lysosomal sialic acid metabolism: retention of sialic acid by Salla disease lysosomes

Purified rat liver lysosomes were incubated in 0.2 M sialic acid resulting in an increase in lysosomal free sialic acid of 3.8 +/- 1.5 nmol/unit beta hexosaminidase. Sialic acid loss by these lysosomes was stimulated 2-3 fold by 25 mM sodium phosphate. Loss of sialic acid by lysosomes from cultured human diploid fibroblasts was similar to that observed in rat liver lysosomes while loss of sialic acid by lysosomes from cultured fibroblasts from a patient with infantile Salla disease occurred much more slowly. Salla disease appears to be the consequence of defective lysosomal transport of sialic acid and is analogous to cystinosis, a disorder of lysosomal amino acid transport.     

N-acetylneuraminic acid accumulation in a buoyant lysosomal fraction of cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured fibroblasts from control individuals and two patients affected with the infantile variant of generalized N-acetylneuraminic acid (NeuAc) storage disease were disrupted by nitrogen cavitation, and the post-nuclear supernatant fractions were subjected to subcellular fractionation on Percoll gradients. Accumulating NeuAc in affected fibroblasts (approx. 150 nmol/mg protein) co-localized with the lysosomal marker N-acetyl-beta-hexosaminidase (Hex), in a fraction with a mean density of 1.035 g/ml. In contrast, more than 70% of the Hex activity of control cells sedimented in comparable gradients with a density of more than 1.07 g/ml. The lysosomal localization of NeuAc accumulation in affected fibroblasts was confirmed by treatment of post-nuclear supernatant fractions with 0.5 mM Gly-Phe-beta-naphthylamide (20 min, 37 degrees C) prior to centrifugation, which resulted in the simultaneous loss of latency of Hex and free NeuAc, and their association with the soluble fraction on Percoll gradients. The results provide direct evidence for the accumulation of free NeuAc in a unique buoyant lysosomal fraction of affected fibroblasts.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.     

Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event

Bacterial cell wall constituents are released from mycobacterial phagosomes and actively traffic within infected macrophages. Colocalization of fluorescently tagged bacterial moieties with endocytic tracers revealed the dynamic movement of released mycobacterial constituents into the endocytic network with accumulation in tubular lysosomal-like compartments. The released bacterial constituents not only penetrated the infected host cell but were also present in an extracellular microvesicular fraction. To identify the intracellular source of these exocytic compartments, released vesicular material was isolated from culture supernatants by differential ultracentrifugation and characterized by Western blot and electron microscopy analyses. The presence of lysosomal membrane proteins and lysosomal proteases suggested that labeled mycobacterial cell wall constituents access a constitutive lysosomal exocytic pathway. An abundance of multilamellar extracellular compartments morphologically reminiscent of MHC class II-enriched compartments (MIIC) implicated a MHC class II transport pathway in the extracellular release of bacterial constituents. Increases in intracellular free calcium have previously been shown to trigger lysosomal exocytosis by inducing fusion of lysosomes with the plasma membrane. To test if an increase in calcium would stimulate exocytosis with release of mycobacterial constituents, infected macrophages were exposed to the calcium ionophore A23187. The ionophore triggered the release of a microvesicular fraction containing labeled bacterial moieties, implicating calcium-regulated lysosomal exocytosis as a trafficking pathway by which mycobacterial products are released from infected macrophages.     

A di-N-acetylchitobiase activity is involved in the lysosomal catabolism of asparagine-linked glycoproteins in rat liver

A perfused rat liver was used to study the effects of 5-diazo-4-oxo-L-norvaline on lysosomal glycoprotein catabolism. Addition of this compound (1.0 mM) to the perfusate reduced activity of beta-aspartyl-N-acetylglucosylamine amidohydrolase by 99% in 1 h. Treated livers were unable to completely degrade endocytosed N-acetyl[14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein as evidenced by a 50% reduction in radiolabeled serum glycoprotein secretion compared to controls. This decreased degradation was matched by a lysosomal accumulation of glycopeptides with the structure: GlcNAc beta(1-4)GlcNAc-Asn. The result suggested the presence of an unrecognized glycosidase in rat liver lysosomes, since this remnant was extended by one more GlcNAc residue than would have been expected after specific inactivation of the amidohydrolase. Such a novel enzyme would therefore catalyze cleavage of the N-acetylglucosamine residue at the reducing end of alpha 1-acid glycoprotein oligosaccharides only following removal of the linking Asn. The activity was then detected in lysosomal extracts by using intact asialo-biantennary oligosaccharides labeled with [3H] galactose or N-acetyl[14C]glucosamine residues as a substrate. To prevent simultaneous digestion of the material from its nonreducing end, beta-D-galactosidase in the enzyme extract was first inactivated with the irreversible active site-directed inhibitor, beta-D-galactopyranosylmethyl-p-nitrophenyltriazene. The observed di-N-acetylchitobiose cleaving activity worked optimally at pH 3.4 and was uniquely associated with the lysosomal fraction of the liver homogenate. The enzyme also cleaved triantennary chains and di-N-acetylchitobiose, but failed to hydrolyze substrates that had been reduced with NaBH4. The new glycosidase was well separated from N-acetyl-beta-D-glucosaminidase (assayed with p-nitrophenyl-beta-D-glucosaminide) by gel filtration chromatography and had an apparent molecular weight of 37,000. A similar enzyme that hydrolyzes di-N-acetylchitobiose had previously been found in extracts of human liver (Stirling, J. L. (1974) FEBS Lett. 39, 171-175).     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Autophagy and lysosomes in Pompe disease

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, intralysosomal glycogen accumulates in multiple tissues, with skeletal and cardiac muscle most severely affected.(1) Complete enzyme deficiency results in rapidly progressive infantile cardiomyopathy and skeletal muscle myopathy that is fatal within the first two years of life. Patients with partial enzyme deficiency suffer from skeletal muscle myopathy and experience shortened lifespan due to respiratory failure. The major advance has been the development of enzyme replacement therapy, which recently became available for Pompe patients. However, the effective clearance of skeletal muscle glycogen, as shown by both clinical and preclinical studies, has proven more difficult than anticipated.(2-4) Our recent work published in Annals of Neurology(5) was designed to cast light on the problem, and was an attempt to look beyond the lysosomes by analyzing the downstream events affected by the accumulation of undigested substrate in lysosomes. We have found that the cellular pathology in Pompe disease spreads to affect both endocytic (the route of the therapeutic enzyme) and autophagic (the route of glycogen) pathways, leading to excessive autophagic buildup in therapy-resistant skeletal muscle fibers of the knockout mice.     

Fluorescence characterization of the low pH-induced change in diphtheria toxin conformation: effect of salt

When injected into rats, a certain amount of mannitol is taken up by the liver and is associated with sedimentable structures. Isopycnic centrifugation in a sucrose gradient shows that a large part of mannitol is present in mitochondria, what remains is located in the lysosomes. The hypotonic release of mannitol present in organelles shows that the polyol is shared between mitochondria and lysosomes. The trapping of mannitol in lysosomes could result from the heterophagic or autophagic function of the lysosomes; the mechanism of its accumulation in mitochondria is still unexplained.     

Cellular ultrastructure of the hepatic sinusoid in partially hepatectomized rats

In male Wistar rats weighing 160-200 g 2/3 of the liver tissue was removed. As a result the phase modifications of lysosome structures in Kupffer's cells have been observed. 2.5 hours after operation the number of primary lysosomal granules increased, 9 hours later an augmentation in size and polymorphism of lysosomes was revealed. At the moment of hepatocyte mitotic peak, i. e. 30 hours after partial liver removal mainly secondary lysosomes were detected in Kupffer's cells. On the contrary, 48 hours following operation the number of new wave of accumulation of primary lysosomal granules was seen. In endothelial cells the lipid infiltration was prevalent especially at the hepatocyte mitotic peak period. The data obtained indicate specific relationship of ultrastructural modifications in sinusoidal cells and phases of the liver reparative regeneration.