Role Of Lysosomal Enzymes Research Articles

Lysosomal enzymes in serum and synovial fluid in patients with osteoarthritis

Objective. To assess the activity of arylsulfatase (AS), acid phosphatase (ACP), cathepsin D (CAT-D) and alpha-1 antitrypsin (AAT) in blood serum and synovial fluid (SF) of patients with hip or knee osteoarthritis (OA). Methods. The study included 43 subjects with OA (35 hip OA, 8 knee OA), submitted total joint replacement. The control group consisted of 58 subjects with no past history of musculoskeletal disorders. Results. The OA blood serum samples showed a significantly higher level of lysosomal enzymes activity than in the control group (AS by 17.8%, AAT by 42.4%); only the CAT-D activity decreased by 50%). AS, ACP and CAT-D activities were about two-fold higher in SF when compared with blood of OA patients. The differences between the genders were visible in the SF: Total protein concentration, activity of ACP (both higher in OA men) and activity of CAT-D (higher in OA women). Between the involved hip and knee, there were no significant differences in all estimated parameters in the blood serum of the OA group. In regard to the SF, only ACP activity was significantly higher in patients with a hip involved. Conclusions. The osteoarthritic SF enzymatic profile differs from that in normal joints. The OA in joints is not reflected in the systemic response. Our preliminary results suggest further studies on role of lysosomal enzymes (ACP and AS) as biomarkers for the detection of osteoarthritis.

Lysosomal enzymes in rat sarcomas induced by 7,12-dimethylbenz(α)anthracene and Rous sarcoma virus

The lysosomal apparatus in normal rat fibroblast cultures and in DMBA and RSV induced rat sarcoma cell cultures was compared. Both DMBA and RSV induced sarcoma cells showed relative increase of lysosomal enzyme activity outside the cells in the medium compared to the activity within the cells. An early equilibrium of enzyme activity level was reached in the medium from RSV sarcoma cells, whereas this was not seen in cultures of normal fibroblasts and DMBA sarcoma cells. There was a difference in the intracellular content of lysosomal enzymes between RSV and DMBA sarcoma cells. This was most clearly seen for N-acetyl-β-glucosaminidase. These findings and the possible role of lysosomal enzymes in malignant cells are discussed.

Effect of cyclophosphamide treatment on selected lysosomal enzymes in the kidney of rats

The anti-cancer drug cyclophosphamide (CYP) is nephrotoxic besides being urotoxic thereby limiting its clinical utility. Since the nephrotoxicity of CYP is less common compared to its urotoxicity, not much importance has been given for the study of mechanism of CYP-induced nephrotoxicity. The aim of the present study is to investigate the possible role of lysosomal enzymes in CYP-induced renal damage. Adult female Wistar rats weighing 200–250 g were used for the study. The rats were administered single-intraperitoneal injection of CYP at the dose of 150 mg/kg body wt and sacrificed at various time intervals 6, 16 or 24 h after the dose of CYP. The control rats were administered saline alone. Nephrotoxicity was assessed by measuring plasma creatinine and urea and histopathology of the kidney. The kidney was weighed and used for the assay of lysosomal enzymes namely acid phosphatase, β-glucuronidase and N-acetylglucosaminidase and total protein content. Histologically, the CYP-treated rat kidneys showed progressive renal damage with increase in time after treatment. Glomerular nephritis, cortical tubular vacuolization and interstitial edema were observed in the CYP-treated rats. Surprisingly, a significant drastic decrease (instead of an increase) in the activities of lysosomal enzymes was observed in the kidneys of CYP-treated rats at 16 and 24 h as compared with the control. A highly significant increase (270%) in protein content was observed in the kidneys of the CYP-treated rats as compared with the control. Decrease in the activities of lysosomal protein digestive enzymes may contribute to CYP-induced renal damage. The accumulation of abnormal amounts of the protein in the kidney may be due at least in part to defect in lysosomal enzyme activity and contribute to renal damage.

Arylsulfatse B in colorectal cancer.

Arylsulfatase B activity has been determined in 24-hour urine samples from 243 patients with colorectal cancer. Elevated activity of the enzyme was obsereved in 172 out of 243 (71%) patients. Employing Dukes' modified classification of colorectal cancer, urine arylsulfatase B activity was elevated in Dukes' A lesions-1/8 (12%), Dukes' B lesions-24/43 (55%). Dukes' C lesions-89/111 (80%), and Dukes' D lesions-66/81 (82%). Arylsulfatase B activity in urine, when elevated, may be used to follow response to therapy since in those patients with elevated urine arylsulfatase B values who subsequently responded to therapy, the enzyme values became or approached normal. Urine arylsulfatase B activity also correlated with plasma carcinoembryonal antigen (CEA) as a diagnostic indicator of colorectal cancer in 33/46 (71%) patients. In contrast to the urinary findings, arylsulfatase B activity in tumor tissue was elevated in only 24% (27/110) of the specimens of colorectal cancer. It was also found that in a group of 55 patients treated with 5-fluorouracil, all of the 13 patients that showed objective response to therapy had activities of arylsulfatase B in the tumor tissue within the normal range for large bowel mucosa. Nevertheless, 22 to 26 of the 43 patients that did not respond also presented values in the normal range. The roles of lysosomal enzymes in colorectal cancer are discussed.

Presence of acid phosphatase in the epidermis of the regenerating tail of the lizard (Podarcis muralis) and its possible role in the process of shedding and keratinization

AbstractThe activity and distribution of the lysosomal marker enzyme acid phosphatase have been studied in the epidermis during tail regeneration in the lizard Podarcis muralis. This study gives information both on the mechanism of separation of the old from the new epidermal generation and on the involvement of lysosomes in β‐ and α‐keratinization in lepidosaurians. The biochemical analysis shows that the activity of acid phosphatase was higher in the regenerating tail than in the normal tail until the stage of skin shedding at around 25–30 days post‐amputation. The enzyme was present in keratinizing layers of epidermis, and particularly in the shedding layer. The shedding layer followed the outline of the regenerating scales beneath the wound in the epidermis showing that lysosomal enzymes are involved in epidermal shedding. The ultrastructural study showed that acid phosphatase was initially localized within the lysosomes but, with the progress of differentiation, it also appeared free within the cytoplasm of the cornifying cells of the shedding complex and of the β‐keratinizing cells, and in the intercellular space among them. The role of lysosomal enzymes in the shedding process and their involvement in the mechanism of β‐keratinization is discussed. While the process of β‐keratinization has an intracellular and extracellular localization of acid phosphatase, the process of α‐keratinization shows that most acid phosphatase is localized within the cell. Lysosomes and mesos granules containing acid phosphatase were concentrated in the cytoplasm of differentiating mesos cells. Some mesos granules later discharged their contents into the extracellular space. The α‐layer was partially formed in proximal regenerated scales around 30 days post‐amputation. Lysosomes appeared to concentrate within pre‐keratinizing cells of the α‐keratin layer and little reactivity was seen extracellularly.

The role of lysosomal enzymes, in particular cathepsin B, in cartilage proteoglycan breakdown within a bovine model

The role of lysosomal enzymes, in particular cathepsin B, in cartilage proteoglycan breakdown within a bovine model

Role of lysosomal enzymes released by alveolar macrophages in the pathogenesis of the acute phase of hypersensitivity pneumonitis

Hydrolytic enzymes are the major constituents of alveolar macrophages (AM) and have been shown to be involved in many aspects of the inflammatory pulmonary response. The aim of this study was to evaluate the role of lysosomal enzymes in the acute phase of hypersensitivity pneumonitis (HPs). An experimental study on AM lysosomal enzymes of an HP-guinea-pig model was performed. The results obtained both in vivo and in vitro suggest that intracellular enzymatic activity decrease is, at least partly, due to release of lysosomal enzymes into the medium. A positive but slight correlation was found between extracellular lysosomal activity and four parameters of lung lesion (lung index, bronchoalveolar fluid total (BALF) protein concentration, BALF LDH and BALF alkaline phosphatase activities). All the above findings suggest that the AM release of lysosomal enzymes during HP is a factor involved, although possibly not the only one, in the pulmonary lesions appearing in this disease.

SERUM CATHEPSIN-B LEVELS AND URINARY-EXCRETION OF CATHEPSIN-B IN THE PATIENTS WITH COLORECTAL-CANCER - POSSIBLE INDICATORS FOR TUMOR MALIGNANCY

Serum cathepsin B levels and urinary excretion of cathepsin B in patients with colorectal cancers were significantly higher than those in the control non-cancer patients. Moreover, cancer tissue cathepsin B content was significantly higher than that in the normal tissue. After radical curative operations for colorectal cancers, both the serum cathepsin B levels and urinary excretion of cathepsin B were restored to the control values. These results suggest a possible role of lysosomal enzyme, cathepsin B, in the pathogenesis of tumor growth, and also suggest that these parameters might be possible indicators for tumor malignancy.

Serum cathepsin B levels and urinary excretion of cathepsin B in the cancer patients with remote metastasis

Serum cathepsin B levels and urinary excretion of cathepsin B in the cancer patients without remote metastasis were significantly higher than those in the control non-cancer patients. Moreover, these parameters in the cancer patients with remote (liver or lung) metastasis were significantly higher than those in the cancer patients without remote metastasis. After radical curative operations, these parameters were restored to the control values. These results suggest a possible role of lysosomal enzyme, cathepsin B in the pathogenesis of tumor metastasis, and also suggest that these parameters might be possible indicators for tumor malignancy such as remote metastasis.

Aggressive role of lysosomal enzymes in gastric ulceration

Administration of lysosomes and lysosome enzymes into the submucous base of intact rats' stomachs did not result in ulceration. Premedication by histamine, serotonin (combined and separated) and ACTH did not increase sensibility of mucous membrane to lysosome enzymes. Prednisolone-premedication provoked the decrease of mucous membrane resistance to free lysosome enzymes and appearance of ulcerative lesions in the point of enzyme applications. Free lysosome enzymes are able to produce ulcerative lesions. This process is going on with the participation of prednisolone.

A New Protease Inhibitor, Nafamostat Mesilate (FUT-175), Protects Pancreatic Acinar Cells in CDE-Diet-lnduced Pancreatitis in Mice

This study was designed to evaluate the role of lysosomal enzymes in the pathogenesis of acute pancreatitis and also evaluate the protective effects of a new synthetic protease inhibitor, naf amostat mesilate (FUT-175), against pancreatic injuries in mice with severe acute pancreatitis induced by a cho-line-deficient ethionine-supplemented (CDE) diet. The CDE diet induced a significant redistribution of cathepsin B activity from the lysosomal fraction (about 30% of the total activity) to the zymogen fraction (about 50%) as compared with the control mice (about 50 and 30%, respectively), which indicated a colocalization of lysosomal enzymes with digestive enzymes in the same sub-cellular compartment. The CDE diet also caused a significant activation of trypsinogen in the pancreatic tissue (14 ± 3 U/mg DNA at 24 h and 21 ± 4U/mg DNA at 48 h). However, FUT-175 exerted significant protective effects against pancreatic injuries induced by CDE diet such as hyperamylasemia (15 ± 2 U/ml at 24 h and 10 ± 3 U/ml at 48 h), high mortality rate (17% at 3 days, 30% at 4 days and 43% at 5 days) as compared with only CDE diet mice (29 ± 3 U/ml at 24 h and 22 ± 3 U/ml at 48 h; 43% at 3 days, 57% at 4 days and 70% at 5 days, respectively). FUT-175 also significantly inhibited the redistribution of cathepsin B activity and activation of trypsinogen (7 ± 2 U/mg DNA at 24 h and 9 ± 2 U/mg DNA at 48 h). These results suggest that lysosomal enzyme, particularly cathepsin B, seems to play an important role in the development of severe acute pancreatitis and that protease inhibitors such as FUT-175 may be clinically beneficial in the treatment of acute pancreatitis.

Role of lysosomal enzymes in drug-induced ulcerogenesis

Role of lysosomal enzymes in drug-induced ulcerogenesis

Role of lysosomal enzymes in tissue damage during hepatic amoebiasis. An experimental study.

Two key lysosomal enzymes, N-acetyl-beta-glucosaminidase and beta-glucuronidase of Kupffer cells and blood monocytes were studied in guinea pigs infected intramesenterically with E. histolytica. The animals were sacrificed on days 0, 2, 5 and 8 post-infection. The enzyme levels in the cell lysate and supernatants of both Kupffer cells and blood monocytes were significantly higher (P less than 0.01) in the infected animals compared with the controls. This increase was more marked on the 5th and 8th days post infection (P less than 0.001). The alteration in enzyme levels was higher in Kupffer cells than in blood monocytes (P less than 0.05). The enzyme levels increased exponentially with the severity of the hepatic lesions. A direct correlation was observed between the enzyme levels and the severity of the hepatic lesions (P less than 0.01). Hence, the role of lysosomal enzymes, released by activated mononuclear phagocytic cells, in the pathogenesis of hepatic amoebiasis is postulated.

Mechanisms of human neutrophil‐mediated cartilage damage in vitro: The role of lysosomal enzymes, hydrogen peroxide and hypochlorous acid

Cartilage is a focal point of attack by cellular and molecular elements of the inflammatory response which occurs in arthritic diseases. Neutrophils damage articular cartilage by degrading matrix components and inhibiting their synthesis. The aim of this study was to elucidate mechanisms of this damage. Human neutrophils were isolated from blood by centrifuging through Ficoll-Hypaque and granule extract prepared from them. Articular cartilage from adult humans and cattle was maintained in organ culture. Cartilage degradation (release of 35S-labelled proteoglycan) or synthesis (incorporation of 35S into proteoglycan) was determined after various treatments. Human neutrophils and neutrophil granule extract degraded proteoglycan and inhibited proteoglycan synthesis. The specific leucocyte elastase inhibitor N-methoxysuccinyl-(ala)2-pro-val-chloromethylketone (MAAPVCMK) partially reversed these effects. H2O2, a product of the neutrophil respiratory burst, when added directly at 10(-6)mol/L, or generated by glucose oxidase (GO)/glucose inhibited proteoglycan synthesis but had no effect on degradation. Hypochlorous acid (OHCl), a product of the myeloperoxidase (MPO)/H2O2/Cl system at 50 mumol/L degraded proteoglycan and inhibited its synthesis. OHCl produced by granule extract (as a source of MPO) + GO-generated H2O2 + Cl- degraded proteoglycan. The results indicate that neutrophil-mediated proteoglycan degradation and inhibition of synthesis is largely attributable to elastase and secondarily to OHCl, whereas H2O2 impairs synthesis without affecting degradation of proteoglycan.

Constitutive secretion of acid hydrolases in Tetrahymena thermophila.

THE role of lysosomal enzymes in intracellular digestion is now well established [11]. Most often we think of lysosomal hydrolases in catabolism of endogenous or foreign material taken up by endocytosis. There is however, a number of reports dealing with the release of acid hydrolases into the extracellular fluid in a variety of eukaryote cells. These cells range from Saccharomyces cerevisiae [15], Dictyostelium discoideum [10], Leishmania donovani [20], Acanthamoeba castellani [22], Entamoeba histolytica [12, 31], and species of Tetrahymena [1–3, 6] to mammalian cells in culture [49]. Concerning the latter, fibroblasts and hepatocytes in culture release acid hydrolases to the extracellular medium, but only if the synthesis of a specific recognition marker is impaired in the cells. This marker (man‐nose‐6‐phosphate) is used for receptor mediated segregation of lysosomal enzymes into the lysosomal compartments. If the receptor or the marker are lacking, the hydrolases fail to enter the lysosomal compartment, and are secreted in immature form together with molecules belonging to the constitutive secretory pathway of the cells [8, 49]. Such a release of acid hydrolases seems to occur spontaneously from mammalian osteoclasts [4]. Macrophages, on the other hand, need a specific stimulation for their release process [40]. In lower eukaryotes the release may

The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole.

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.

Midgut Endocrine Cells in the Mosouito, Aedes Aegypti

The ultrastructure of a cell type in the insect midgut resembles that of peptide hormonogenic cells in the vertebrate gut. Also, vertebrate-like peptides such as pancreatic polypeptide (PP), glucagon, and insulin have been detected by immunoassays in insect midguts. Research was initiated to characterize the endocrine cells in the mosquito midgut, determine their immunoreactivity to antisera against vertebrate peptide hormones, and to identify organelles related to secretion of the hormones.Female mosquitos had access to water or a sucrose solution for 7 days. Midguts from the insects were processed for TEM by routine methods. To assess the role of lysosomal enzymes in the secretory process, midguts were incubated in cerium-based medium for acid phosphatase histochemistry. For immuno-cytochemistry, midguts were fixed in a modified Bouin's solution (pH 7.6) and 156 glutaraldehyde for 1 hr and embedded in epon/araldite. Thin sections of midgut on nickel grids were sequencially emersed in filtered rabbit antisera to PP, 1:1000 in 0.05M Tris (pH 7.6); anti-rabbit IgG, 1:20; and PAP, 1:200.

A role of lysosomal enzymes in the mechanism of mucolytic action of bromhexine.

The effect of bromhexine on behaviors of lysosomal enzymes in the submucosal gland was investigated using canine tracheal slice preparations, with reference to the histochemical changes in acid glycoproteins (AGP) in the gland. Incubation of tracheal slices with 1% Triton X-100 or 0.0004-0.04% bromhexine for 30 min decreased the number of stained lysosomes in the glandular cells. The decrease in stained lysosomes after treatment with 1% Triton X-100 or 0.04% bromhexine was effectively prevented by addition of 5% lecithin. The number of glandular cells that were stained red (stain index R) in the combined alcian blue at pH 2.5 and periodic acid-Schiff procedure markedly increased by treatment with 1% Triton X-100 or 0.0004-0.04% bromhexine. In the bromhexine treated groups, there was a close correlation (r=0.932, P less than 0.01) between the increase in the number of glandular cells showing the stain index R and the decrease in the number of stained lysosomes in the cells. These findings suggest that the enzymes which are liberated from lysosomes into the cytoplasm by bromhexine may, at least in a part, be involved in the mucolytic action of the agent on AGP contained in mucus granules of the submucosal gland.

Lysosomal enzymes in ocular tissues and diseases

Lysosomal enzymes are distributed widely in various ocular tissues. Among these tissues, the uvea and retina show the higher enzyme activities of acid phosphates, β-blucuronidase, α-fucosidase, α-mannosidase, arylsulfatase, cathepsin D, cathepsin B and others. The particular role of lysosomal enzymes in the pathogenic processes of ocular diseases such as storage disease, uveitis, retinal degeneration, retinal detachment, corneal dystrophy and glaucoma is strongly suggested. The enzymes also have additional importance in ocular physicopathology.

Effects of gamma radiation on rabbit lens.

Eyes of young New Zealand white rabbits were irradiated with 2000 rads of gamma radiation (60Co) and the eyes removed at 2, 8, 16, and 30 days post-irradiation for electron microscopic analysis. Lenses were treated histochemically for acid phos-phatase localization to examine the role of lyso-somal enzymes in the early development of radiation cataracts. Progressive degenerative morphological changes due to radiation included pronounced nuclear degeneration and cellular shrinkage within the germinative and pregerminative epithelium and severe swelling of superficial portions of lens fibers beneath these epithelial zones. Less dramatic changes occurred elsewhere in the superficial portions of the anterior and marginal lens segments. The epithelium of both irradiated and non-irradiated lenses contained small (60-70 nm) vesicles fused with the apical and lateral cellular borders. Irradiated lenses showed a 30-40% increase in population of these vesicles at all time intervals in the central (including the pre...