Synthesis Of Lysosomal Enzymes Research Articles

α-Synuclein dimerization in erythrocytes of Gaucher disease patients: correlation with lipid abnormalities and oxidative stress

Several observations suggest that disturbed homeostasis of α-Synuclein (α-Syn) may provide a link between Gaucher disease (GD) and Parkinson’s disease (PD). We recently reported increased dimerization of α-Syn in the red blood cell (RBC) membrane of patients with GD. Several studies indicate a crucial relationship between lipids, oxidative stress and α-Syn status. Here we investigated the relationship between the observed increased dimerization of α-Syn in the cell membranes of RBCs, cells devoid of lysosomes and lacking lysosomal enzyme synthesis, and the lipid abnormalities and oxidative stress already described in GD. Correlation studies showed that in GD the α-Syn dimer/monomer ratio is positively correlated with the levels of glucosylceramide (GlcCer) and the glucosylceramide/ceramide (GlcCer/Cer) ratio and negatively with the levels of malonyldialdehyde (MDA) and plasmalogens. In conclusion, we have shown that the increased tendency of α-Syn to form dimers in the RBC membrane of patients with GD, is correlated with both the level of lipids, including GlcCer, the primary lipid abnormality in GD, and the increased oxidative stress observed in this disorder. The study of other tissues, and in particular brain, will be important in order to elucidate the significance of these findings regarding the link between GD and PD.

Palate development. In vitro procedures.

The mammalian secondary palate provides an excellent experimental paradigm with which to investigate regulation of the sequential expression and interaction of molecular signals during embryogenesis. The secondary palate arises as bilateral extensions of the oral aspect of the maxillary processes of the first arch. In mammalian embryos, these palatal extensions make contact, fuse with one another and give rise to the secondary palate (roof of the oral cavity) (,). The palatal processes, consisting of mesenchymal cells embedded in a loosely organized extracellular matrix and enclosed within a stratified epithelium, initially grow vertically on either side of the tongue. The processes subsequently undergo a series of morphogenetic movements that result in their reorientation above the tongue, thereby bringing the medial edge epithelium (MEE), on the apical surface of each process, into contact forming a midline epithelial seam. Cells of the MEE undergo a precise sequence of molecular changes that culminate in removal of the midline epithelial seam. These changes include cessation of DNA synthesis, increased synthesis of cell-surface glycoconjugates, increased synthesis of lysosomal enzymes, cell death of the peridermal (surface) cell layer, and transdifferentiation of the subjacent MEE from an epithelial to a mesenchymal phenotype. Fusion of the palatal processes, followed by peridermal cell death and transdifferentiation of the MEE and migration into the underlying mesenchyme results in formation of the definitive secondary palate (,).

Isolation and characterisation of pig Peyer's patch dendritic cells

We have isolated dendritic cells (DC) from Peyer's patches (PP) of pig small intestine by mechanical tissue disruption followed by fractionation of isolated cells on metrizamide gradients. Characterisation was carried out using the following criteria: morphology; lysosomal enzyme synthesis; expression of membrane antigens; and capacity for antigen presentation. Dendritic cells did not express acid phosphatase or β-galactosidase, but were weakly positive for non-specific esterase and ATPase. Dendritic cells did not express CD3, CD2, sIg, or an antigen specific for pig mononuclear phagocytes and granulocytes. They did, however, express MHC class II at very high levels. They were shown to be potent stimulators in an allogeneic mixed lymphocyte reaction.

Effects of ethanol and protein deficiency on pancreatic digestive and lysosomal enzymes.

The pathogenesis of alcoholic pancreatitis is not fully understood. An increase in pancreatic digestive and lysosomal enzyme synthesis because of ethanol consumption could contribute to the development of pancreatic injury in alcoholics. This study aimed, firstly, to determine the effect of ethanol on the content and messenger RNA levels of pancreatic digestive enzymes and on the messenger RNA level of the lysosomal enzyme cathepsin B, and secondly, to examine the influence of concomitant protein deficiency (a known association of alcoholism and pancreatic injury) on these effects. A rat model of chronic ethanol administration was used in which rats were fed in groups of four, and for four weeks, protein sufficient and protein deficient diets with or without ethanol. Ethanol increased the pancreatic content of lipase but did not influence chymotrypsinogen or trypsinogen values. mRNA levels for lipase, trypsinogen, and chymotrypsinogen were raised in rats fed ethanol. Protein deficiency resulted in reduced tissue levels of lipase, chymotrypsinogen, and amylase but did not influence trypsinogen values. mRNA levels for proteases were increased in protein deficient rats, while those for lipase remained unaltered. Both ethanol and protein deficiency increased mRNA levels for cathepsin B. It is concluded that chronic ethanol consumption, in both protein sufficient and protein deficient states, increases the capacity of the pancreatic acinar cell to synthesise digestive and lysosomal enzymes.

Molecular mechanisms of bone resorption by the osteoclast

The osteoclast is a multinucleated cell that is actively engaged in the synthesis of lysosomal enzymes, their vectorial transport toward the apical membrane, and the secretion of these enzymes at its apical pole. These secreted enzymes are targeted to the apical ruffled-border membrane by mechanisms that involve cation-independent mannose-6-phosphate receptors. These receptors bind to an enzyme-linked mannose-6-phosphate recognition marker in the Golgi complex, and the enzyme-ligand-receptor complex, carried within small coated transport vesicles, dissociates upon reaching the low pH established in the bone-resorbing compartment by the osteoclast. The apical bone-resorbing compartment is sealed off by the attachment of the osteoclast to the calcified matrix and is actively acidified by the osteoclast. The plasma membrane of the cell is divided into distinct domains. The apical membrane at the ruffled-border shares common antigenic determinants with lysosomal and endosomal membranes, including a 100 kD protein and proton pumps that may be involved in the acidification of the extracellular resorbing compartment. The basolateral membrane is highly enriched in carbonic anhydrase, and bicarbonate-chloride exchange appears to regulate the intracellular pH of this cell. These observations are consistent with a scheme in which, in the low pH environment of the bone-resorbing lacuna produced by the osteoclast, the mineral phase dissolves, exposing the organic matrix to the action of the secreted enzymes. The activity of these enzymes is in turn presumably favored by the acidic milieu. All constituents of the matrix, whether mineral or organic, then would be reduced to their elemental forms (ions and amino acids) extracellularly.(ABSTRACT TRUNCATED AT 250 WORDS)

Bactericidal power of neutrophils in protein calorie malnutrition

The present study was undertaken to compare the disturbance in bactericidal power of neutrophils in 25 children having protein caloric malnutrition age and sex matched healthy control subjects. There was a highly significant reduction in the percentage of bacteria killed during incubation in children having PCM as compared to healthy control. A direct relationship existed between total serum proteins and bactericidal activity of neutrophils in children having PCM. The impaired bactericidal power of neutrophils can be attributed to impaired synthesis of lysosomal enzymes, glycolytic activity of neutrophils in children having PCM. The exact mechanisms still remain to be elucidated. Decreased bactericidal activity of neutrophils may be one of the mechanisms responsible for infection.

The effect of intracellular Chlamydia psittaci on lysosomal enzyme activities in mouse peritoneal macrophages.

Live or heat killed (30 min at 56 degrees C) Chlamydia psittaci elementary bodies (EB) were phagocytosed by mouse peritoneal macrophages. Inoculation with killed Chlamydia caused rises in three lysosomal enzyme activities tested, especially in acid phosphatase activity. In contrast, after infection with live Chlamydia, only a negligible increase was seen in acid phosphatase activity, and no change in the activities of beta-glucuronidase and cathepsin D was observed. It was concluded that regulation of lysosomal enzyme synthesis in macrophages may be linked to signals mediated by phago-lysosome fusions.

Contact-induced lymphocyte lysosomal enzyme synthesis and transfer

Contact-induced lymphocyte lysosomal enzyme synthesis and transfer

Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL-60: application to the study of myeloid lysosomal enzyme synthesis and processing

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.

Separation and Analysis of Subcellular Organelles in a Human Promyelocytic Leukemia Cell Line, HL-60: Application to the Study of Myeloid Lysosomal Enzyme Synthesis and Processing

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.

Synthesis and intracellular localization of chick acid α-glucosidase in chick erythrocyte-human fibroblast heterokaryons: A model system for the study of lysosomal enzyme synthesis

The synthesis and localization of chick acid α-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid α-glucosidase were used. It was found that the acid α-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.

The mucopolysaccharidoses: a synergism between clinical and basic investigation.

The mucopolysaccharidoses are a family of genetic diseases involving faulty degradation of one or more type of mucopolysaccharide or glycosaminoglycan. These conditions are characterized by skeletal, connective tissue, and intellectual manifestations. Biochemical investigations over the past 10-20 yr have enhanced our understanding of the clinical conditions and advanced our knowledge of catabolic pathways for complex biological structures. Identification of stored and excreted materials as partially degraded glycosaminoglycans allowed classification of these conditions on clinical and chemical criteria. The development of a cell culture model for studying the mucopolysaccharidoses lead to the identification of a variety of lysosomal sulfatases and glycosidases as deficient enzymes. This stimulated the identification of several new conditions. Knowledge of primary biochemical defects improved genetic services by providing accurate diagnosis, facilitating carrier identification and improving prenatal diagnosis. Glycosaminoglycan excretion patterns associated with individual enzyme defects suggested sequential degradative pathways for GAGs and related macromolecules. Attempts at enzyme replacement revealed a complex system of recognition markers and membrane receptors for the uptake of extracellular proteins. Multiple enzyme deficiencies are providing information on lysosomal enzyme synthesis and processing. The mucopolysaccharide storage diseases offer an excellent example of a productive synergism between basic biology and clinical medicine.

The Mucopolysaccharidoses: A Synergism between Clinical and Basic Investigation

The mucopolysaccharidoses are a family of genetic diseases involving faulty degradation of one or more type of mucopolysaccharide or glycosaminoglycan. These conditions are characterized by skeletal, connective tissue, and intellectual manifestations. Biochemical investigations over the past 10-20 yr have enhanced our understanding of the clinical conditions and advanced our knowledge of catabolic pathways for complex biological structures. Identification of stored and excreted materials as partially degraded glycosaminoglycans allowed classification of these conditions on clinical and chemical criteria. The development of a cell culture model for studying the mucopolysaccharidoses lead to the identification of a variety of lysosomal sulfatases and glycosidases as deficient enzymes. This stimulated the identification of several new conditions. Knowledge of primary biochemical defects improved genetic services by providing accurate diagnosis, facilitating carrier identification and improving prenatal diagnosis. Glycosaminoglycan excretion patterns associated with individual enzyme defects suggested sequential degradative pathways for GAGs and related macromolecules. Attempts at enzyme replacement revealed a complex system of recognition markers and membrane receptors for the uptake of extracellular proteins. Multiple enzyme deficiencies are providing information on lysosomal enzyme synthesis and processing. The mucopolysaccharide storage diseases offer an excellent example of a productive synergism between basic biology and clinical medicine.

Effect of an aromatic retinoid on human squamous cell carcinomas in vitro

After in vitro incubation of human squamous cell carcinomas of the head and neck region with aromatic retinoid an increased number of lysosomes can be observed in the tumor cells. It is discussed whether this accumulaion of lysosomes is due to a direct stimulation of lysosomal enzyme synthesis or whether it is consequence of cell damage by the retinoid.

Lysosomal dysfunctions associated with mutations at mouse pigment genes.

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.

Elevation of plasma levels of lysosomal enzymes during treatment with rifampicin and isoniazid

The activities of the enzymes γ-glutamyl transpeptidase, β-glucuronidase, β- N-acetylglucosaminidase, and alanine transaminase were measured in the plasma of 24 patients receiving treatment for tuberculosis with rifampicin and isoniazid. Except for seven patients, five of whom were known to be high alcohol consumers, γ-glutamyl transpeptidase levels were normal and did not reflect the known inducing effects of rifampicin on liver cell microsomal enzymes. The predominantly lysosomal enzymes β-glucuronidase and β- N-acetylglucosaminidase were significantly raised compared to controls (p < 0.001), and there was a significant correlation between the two enzymes ( r = 0.661, p < 0.001). This elevation occurred within 4 weeks and was then independent of the duration of treatment. Alanine transaminase levels were normal, excluding any more general hepatocellular damage. These findings suggest an increase in lysosomal enzyme synthesis and release during rifampicin and isoniazid therapy.

Concanavalin A and the in vitro induction in macrophages of vacuolation and lysosomal enzyme synthesis

Concanavalin A (ConA) induced extensive vacuolation in mouse peritoneal macrophages. Electron microscopic observations on thin sections reveal that the vacuoles are essentially empty except for minute vesicles attached to their inner periphery. The vacuoles consist of irregular structures and are heterogeneous in size distribution. ConA-induced vacuoles exhibit high acid phosphatase activity, suggesting fusion between vacuoles and lysosomes. Induction of acid phosphatase in ConA-treated macrophages was studied under several cultivation conditions. ConA-treated macrophage cultures responded in increase in acid phosphatase activity early after exposure to the lectin, a significant increase recorded already after 1 h. When cultivated in 1% serum medium for 48 h, ConA-treated macrophages exhibit twice the activity of acid phosphatase at zero time as well as that of non-treated control cultures. The effect of ConA on thioglycolate-stimulated mouse peritoneal macrophages was also studied. Vacuole formation resulting from lectin binding and internalization is discussed in terms of possible lectin effects on membrane fluidity, fusion capacity, surface to volume conservation during vacuole formation, fusion of vacuoles with lysosomes and intravacuolar lysosomal enzyme activities. The phenomenon of lysosomal enzyme induction as a result of ConA treatment is being correlated with enzyme induction due to other stimuli.

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β- N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.

Ultrastructural morphology and histochemistry (acid phosphatase) of the cutaneous infiltration by polyvinylpyrrolidone.

SUMMARY.– Two new cases of skin infiltration by polyvinylpyrrolidone are reported. One had 2 types of macroscopic lesion: erythematous plaques, often with yellow pseudo-xanthoniatous papules in the centre, localized round the neckline and the front of the thorax, and small fibromata on the thighs and legs. The other patient had slightly indurated skin in the epigastric region. The ultrastructure of the histiocyte infiltration consisted of dense bodies and vacuoles with various structural variations. The authors stress the extremely generalized nature of this infiltration, affecting all the cell types of the skin, and the presence of intermediary forms linking the dense bodies to the vacuoles. Acid phosphatase activity is present in the dense bodies and certain intermediary vacuoles but absent from the large vacuoles. It is suggested that the dense bodies represent lysosomes with enzyme accumulation which then become loaded with polyvinylpyrrolidone, thus leading to the formation of large vacuoles with no enzyme activity. The well-developed Golgi apparatus in the histiocytes plays a part in the synthesis of lysosomal enzymes.

A histological and histochemical study of skeletal muscle regeneration in polymyositis

The histological features of regenerating muscle fibres in biopsies from cases of polymyositis have been examined, and the activities of certain enzymes in these fibres have been assessed histochemically. Regenerative changes were most prominent in segments of muscle fibres which had undergone necrosis. Mononucleated myoblasts appeared at the periphery of necrotic fibres before completion of phagocytosis and divided by mitosis. Multinucleated syncitia were found in a similar position and extended along empty sarcolemmal tubes. No evidence of nuclear division was found in these structures, suggesting that they were formed by myoblast fusion. Budding from the remaining ends of necrotic fibres was a less frequent finding. However, changes suggestive of increased ribonucleoprotein formation in the terminal ends of these fibres were commonly found and were considered to be indicative either of regenerative activity or of lysosomal enzyme synthesis. Degenerative changes such as vacuolation and nuclear pyknosis were found in some regenerating fibres. A second biopsy from the same muscle after clinical recovery in one case showed completely normal muscle architecture indicating that complete regeneration had occurred. Regenerating fibres were found to have a high content of RNA and high LDH, SDH and NADH-dehydrogenase activities. However, only small amounts of neutral fat were present and phosphorylase activity was completely absent in regenerating fibres. The present findings demonstrate the ability of the damaged muscle fibre to regenerate completely in polymyositis and contrast with the ineffective regenerative changes found in the Duchenne form of muscular dystrophy. These differences account for the contrasting natural histories of these two myopathies.