Electromyographic activity and noradrenaline content of the rabbit oviduct under different hormonal states

Spontaneous electromyographic (EMG) activity of the oviduct recorded in vivo in untreated, estrogen-treated, and progesterone-treated castrated rabbits was found to exhibit two main patterns: short spike bursts lasting 1-10 s and long trains of action potentials lasting several minutes, which constituted the major component of EMG activity. After estrogen treatment, both wet weight and noradrenaline (NA) content of the castrated rabbit oviduct were enhanced mainly at the ampullary-isthmic junction; long trains of discharges were significantly shorter (2.0-2.7 min vs 3.6-4.6 min) and appeared at more frequent intervals (9.8-12.2 min vs 14.2-22.6 min). After progesterone treatment, spontaneous EMG activity was not significantly different from that in untreated castrated rabbits (as was the NA content) except at the ampullary-isthmic junction. NA injection elicited a stimulatory response of the oviduct lasting 1-7 min in the three hormonal states. Phentolamine strongly depressed spontaneous EMG activity but the inhibition was more transient in castrated rabbits than in estrogen-treated and progesterone-treated animals. Propranolol had no effect on spontaneous EMG activity. These data and the high NA concentrations found in all parts of the isthmus support the hypothesis that adrenergic innervation plays a role in the organization of oviductal motility in the rabbit.     

Glycosidase activity in phenotypically different pathologic lymphoid cells, found at various stages of differentiation

The activities of seven glycosidases (six lysosomal and one cytosolic) were determined in B- and T-lymphoid cells differing by immunological phenotypes and occurring at various differentiation stages. The cells were isolated from the circulating blood, bone marrow or spleens of patients with various forms of lymphoproliferative disorders. The glycosidase activities varied significantly depending on the phenotype. The highest activity of all glycosidases was observed in cells with a common lymphoid cell progenitor phenotype. In cells having the phenotype of mature T- and B-cells the glycosidase activities were comparatively low. The changes in all glycosidase activities depending on the phenotype and differentiation stage usually occurred in the same direction; however, the degree of elevation or decline of activities of individual glycosidases was different. The activities of N-acetyl-beta-D-hexosaminidase and alpha-D-mannosidase changed dramatically, whereas the changes in the activity of cytosolic neutral alpha-D-glucosidase were less apparent. These data suggest that lysosomal glycosidases play specific roles in lymphoid cell differentiation.     

Purification and immunological quantification of rat liver lysosomal glycosidases.

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.     

High levels of GM1-ganglioside beta-galactosidase in the salivary glands and GM1-like-ganglioside storage in parotids of deficient mice.

We have previously demonstrated high levels of GM1-ganglioside beta-galactosidase (beta-gal) in the salivary glands of Swiss-Webster mice (Nowroozi et al., J Craniofac Genet Dev Biol 18:51, 1998), and suggested that this activity reflects an important role for the lysosome in catabolism of salivary glycoconjugates. Here, we characterized and compared activities of lysosomal glycosidases among the salivary glands, spleen, and muscle of C57BL/6 mice, beta-gal hexosaminidase, and beta-glucuronidase activities are high in all three glands relative to muscle. Enzyme activities in the sublingual gland were substantially higher than in the submandibular and parotid glands. Spleen displays levels of activity that are comparable or higher (for beta-glucuronidase) than those in the salivary glands, whereas muscle displays substantially lower levels of these lysosomal glycosidases. In order to investigate the role of beta-gal in the salivary glands, we further characterized the salivary phenotype of knock-out mice deficient in this enzyme, mimicking human GM1-gangliosidosis. In contrast with the relative levels of beta-gal specific-activity among the salivary glands, only the parotid developed severe, generalized, degenerative histopathological changes in beta-gal-deficient knock-out mice. GM1-like-ganglioside, typically found at high levels only in the nerve tissue, where its exact function is still not clear, was demonstrated in storage vacuoles of the parotid glands of the deficient mice by binding of cholera toxin subunit B. Thus, beta-gal activity observed in the parotid gland most likely reflects its role in GM1-ganglioside catabolism, and this ganglioside, never previously reported in the salivary glands, may have a role in parotid exocrine secretory functions. beta-gal may also serve in secretory glycoprotein catabolism in other salivary glands, but this function may be non-essential for these glands.     

Deficient lysosomal carboxypeptidase activity in galactosialidosis

In the lysosome, the glycosidases neuraminidase (EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23) are associated to a 52 kDa "protective protein" to form a large multi-enzymatic complex. Deficient synthesis or inactivation of this protective protein causes galactosialidosis, a lysosomal storage disorder in man in which both neuraminidase and beta-galactosidase activities are deficient. Since the protective protein possesses extensive sequence homology with carboxypeptidase Y (carb Y) and the KEX 1 gene product from yeast, we have used the artificial substrate N-CBZ-Phe-Leu to detect and characterize the peptidase activity of the lysosomal carboxypeptidase (carb L). Using both a purified preparation of the lysosomal multi-enzymatic complex and cultured skin fibroblasts of patients affected with galactosialidosis, we demonstrate that the 52 kDa protective protein is responsible for carb L activity. The fibroblasts of three patients affected with late infantile and juvenile galactosialidosis were found to be deficient in carb L activity (1.4% of normal mean value).     

Lysosomal metabolism of glycoproteins

The lysosomal catabolism of glycoproteins is part of the normal turnover of cellular constituents and the cellular homeostasis of glycosylation. Glycoproteins are delivered to lysosomes for catabolism either by endocytosis from outside the cell or by autophagy within the cell. Once inside the lysosome, glycoproteins are broken down by a combination of proteases and glycosidases, with the characteristic properties of soluble lysosomal hydrolases. The proteases consist of a mixture of endopeptidases and exopeptidases, which act in concert to produce a mixture of amino acids and dipeptides, which are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Although the glycans of all mature glycoproteins are probably degraded in lysosomes, the breakdown of N-linked glycans has been studied most intensively. The catabolic pathways for high-mannose, hybrid, and complex glycans have been established. They are bidirectional with concurrent sequential removal of monosaccharides from the nonreducing end by exoglycosidases and proteolysis and digestion of the carbohydrate-polypeptide linkage at the reducing end. The process is initiated by the removal of any core and peripheral fucose, which is a prerequisite for the action of the peptide N-glycanase aspartylglucosaminidase, which hydrolyzes the glycan-peptide bond. This enzyme also requires free alpha carboxyl and amino groups on the asparagine residue, implying extensive prior proteolysis. The catabolism of O-linked glycans has not been studied so intensively, but many lysosomal glycosidases appear to act on the same linkages whether they are in N- or O-linked glycans, glycosaminoglycans, or glycolipids. The monosaccharides liberated during the breakdown of N- and O-linked glycans are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Defects in these pathways lead to lysosomal storage diseases. The structures of some of the oligosaccharides that accumulate in these diseases are not digestion intermediates in the lysosomal catabolic pathways but correspond to intermediates in the biosynthetic pathway for N-linked glycans, suggesting another route of delivery of glycans to the lysosome. Incorrectly folded or glycosylated proteins that are rejected by the quality control mechanism are broken down in the ER and cytoplasm and the end product of the cytosolic degradation of N-glycans is delivered to the lysosomes. This route is enhanced in cells actively secreting glycoproteins or producing increased amounts of aberrant glycoproteins. Thus interaction between the lysosome and proteasome is important for the regulation of the biosynthesis and distribution of N-linked glycoproteins. Another example of the extralysosomal function of lysosomal enzymes is the release of lysosomal proteases into the cytosol to initiate the lysosomal pathway of apoptosis.     

The synthesis of prostaglandin F by human endometrium in organ culture

Slices of human endometrium obtained from hysterectomy specimens were cultured for 48 hours in an organ culture medium supplemented with ethanol (control, vehicle), 17-β-estradiol (.5 μg/ml), or progesterone (.5 μg/ml). Uncultured endometrial tissue, cultured tissues, and the media were assayed for prostaglandin F (PGF) by a radioimmunoassay technique. Hematoxylin and eosin and periodic acid-Schiff stain histologic controls were done on all tissues. The concentrations of PGF in picograms/milligram, corrected for percent recovery, in the differently treated tissues were: preculture 298; culture control 2210; estrogen-treated 2680; progesterone-treated 1260. All differences except those between estrogen and control (p >; .10) and progesterone and control (p < .10) are significant at the p = .02 level or better. Progesterone appears to inhibit PGF synthesis which occurs during in vitro culture of human endometrium; estrogen tended to increase PGF synthesis in this system.     

Effect of swainsonine on rat epididymal glycosidases

Each epididymis of control and swainsonine-fed rats (5 micrograms/ml drinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosidases and total protein were determined, the proximal cauda contained the greatest concentration of glycosidase. The specific-activity profile for beta-glucuronidase and beta-galactosidase was similar in swainsonine-fed and control rats. However, the concentration of alpha-D-mannosidase in tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for alpha-D-mannosidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the alpha-D-mannosidase activity was optimal at pH 4.5 and studies with highly specific antibody to lysosomal alpha-D-mannosidase immunoprecipitated all of the alpha-D-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free supernatant, the enzyme studied is of lysosomal origin.     

Induction of lysosomal glycosidases by dibutyryl cAMP in neuroblastoma cells

We have studied the regulation of lysosomal glycosidases during morphological differentiation of NB2a neuroblastoma cells. Cells treated with dibutyryl cAMP induced axon-like neurites and showed a 2–4 fold increase in the activity of 6 lysosomal glycosidases, reaching their highest level after 5 days of treatment. Cells treated with retinoic acid, which induced dendrite-like neurites, did not show significant changes in the glycosidases activity although cell proliferation was also inhibited. There was no change in the pattern of the enzyme secretion during the dibutyryl cAMP treatment and morphological analysis using electron microscopy and cytochemical staining with acid phosphatase indicated the presence of lysosomes in the induced neurites.     

Localization of dolichol in the lysosomal fraction of rat liver

The distribution of dolichol and/or dolichol esters in subcellular fractions prepared from a rat liver homogenate has been investigated. After saponification of the various fractions dolichol was isolated and quantitated by high performance liquid chromatography in three systems. The degree of purity of the subcellular preparations was examined by marker enzymes and by electron microscopy. Using differential centrifugation it was found that the level of dolichol was highest in the mitochondria-lysosome fraction. Upon further resolution of this fraction by sucrose density centrifugation it was found that the majority of the dolichol was associated with the lysosome-rich fraction. In contrast, the mitochondrial fraction had only a low level of dolichol. This novel observation was confirmed by the finding that dolichol was greatly enriched in a highly purified lysosome fraction preparations isolated by Metrizamide density centrifugation. The enrichment of dolichol in this purified preparation paralleled the observed enrichment of the lysosomal enzyme activity in this fraction. All of these data suggest that the majority of cellular dolichol and/or dolichol esters is localized in the lysosome fraction. The significance of this finding in relation to the metabolism of dolichol is discussed.     

Lysosomal peptidases and glycosidases in rheumatoid arthritis

Lysosomal serine and cysteine proteases are reported to play a role in collagen degradation. In this study, the activities of the lysosomal cysteine proteases cathepsin B and H, dipeptidyl peptidase I, and the serine protease tripeptidyl peptidase I and dipeptidyl peptidase II, all ascribed a role in collagen digestion, were compared with those of the aspartate protease cathepsin D, and lysosomal glycosidases in leukocytes from rheumatoid arthritis patients at different stages of the disease. In all patients the activities of cysteine protease cathepsin B, dipeptidyl peptidase I, aspartate protease cathepsin D, and two glycosidases were elevated, but the activities of the serine proteases tripeptidyl peptidase I, dipeptidyl peptidase II, and the cysteine protease cathepsin H was unchanged. The magnitude of the increased activity was correlated with the duration of the disease. Patients with long-standing RA (10 years or more) had higher cysteine protease activity in their leukocytes than did those with disease of shorter duration. This tendency suggests that elevated lysosomal cysteine protease activities, together with aspartate protease cathepsin D and lysosomal glycosidases (but not serine proteases), are associated with progression of rheumatoid arthritis.     

mTOR activates the VPS34–UVRAG complex to regulate autolysosomal tubulation and cell survival

Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient‐sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome‐lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3‐phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3‐kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.     

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.     

Lysosomal involvement in the removal of clofibrate-induced rat liver peroxisomes. A biochemical and morphological analysis

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate-treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug-induced peroxisomes was investigated under physiological conditions, ie in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal β-oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.     

Selective Yolk Deposition and Mannose Phosphorylation of Lysosomal Glycosidases in Zebrafish

The regulation and function of lysosomal hydrolases during yolk consumption and embryogenesis in zebrafish are poorly understood. In an effort to better define the lysosomal biochemistry of this organism, we analyzed the developmental expression, biochemical properties, and function of several glycosidases in zebrafish eggs, embryos, and adult tissues. Our results demonstrated that the specific activity of most enzymes increases during embryogenesis, likely reflecting a greater need for turnover within the embryo as yolk-derived nutrients are depleted. Analysis of glycosidase activity in zebrafish and medaka eggs revealed selective deposition of enzymes required for the degradation of N-linked glycans, including an abundance of acidic mannosidases. Treatment of zebrafish embryos with the α-mannosidase inhibitor swainsonine resulted in the accumulation of glycosylated vitellogenin fragments and demonstrated a function for maternally deposited acid α-mannosidase in yolk consumption. Surprisingly, we also found that, unlike mammals, acid α-glucosidase from zebrafish and medaka does not appear to be modified with mannose 6-phosphate residues. We further showed these residues were not acquired on human acid α-glucosidase when expressed in zebrafish embryos, suggesting unique differences in the ability of the human and zebrafish N-acetylglucosamine-1-phosphotransferase to recognize and modify certain lysosomal glycosidases. Together, these results provide novel insight into the role of acidic glycosidases during yolk utilization and the evolution of the mannose 6-phosphate targeting system in vertebrates.     

Alterations in the heart lysosomal stability in isoproterenol induced myocardial infarction in rats

The alterations in the heart lysosomal stability following isoproterenol induced myocardial infarction were studied in albino rats. The rate of release of beta-glucuronidase at various time intervals at 37 degrees C from lysosome rich fraction was taken as a measure of lysosomal stability. As compared to the control day one, three and five samples exhibited a significant increase in beta-glucuronidase activity at all the time intervals. The subcellular distribution of beta-glucuronidase was also studied and the soluble and total activities exhibited an increase at peak infarction stage and returned to normal during the recovery. The decrease in the lysosomal stability might be attributed to the increased beta-glucuronidase activity observed following myocardial infarction.     

Rupture of rat liver lysosomes mediated by l-amino acid esters

Treatment of rat liver lysosome suspensions with 0.5–20 mM α-l-amino acid esters results in a progressive loss of latency of lysosomal enzyme activity. The increase in available acid phosphatase activity in lysosomal suspensions is correlated with the decrease of turbidity of these suspensions. Ester mediated turbidity decrease is dependent upon ester concentration, and the pH and ionic strength of the suspending medium. d-Stereoisomers of amino acid esters do not exhibit comparable capacity to damage lysosomes.α-l-Amino acid esters were found to be substrates for neutral lysosomal esterase and transpeptidase activity. The d-stereoisomers are degraded at much lower rates. These data support the hypothesis that ester dependent lysosomal rupture is mediated by the specific interaction of the ester with a structural or functional lysosomal protein.     

Lysosome rich cells contain the lytic activity of lymphokine-activated killer cell populations

Summary Little is known regarding the effectors of lymphokine-activated killer activity. Lysosomotropic agents such as quinacrine can be used to positively sort for lysosome rich cells in natural killer (NK) cell populations. We therefore decided to use this agent to sort lymphokine-activated killer (LAK) cells to characterize their lysosomal content. We found that the positively sorted population contained all the LAK activity, i.e., lysis of NK-resistant tumor cells (B16 melanoma cell line), with the negatively sorted cells having no killing activity. Therefore separation of interleukin-2-incubated cells for LAK activity could be accomplished using sorting after quinacrine staining. The treatment of positively sorted LAK cell populations with L-leucine methyl ester, a lysosomotropic dye which inhibits killing by lysosome rich cells, caused abrogation of killing of the B16 tumor by the treated populations. Single cell conjugate assays were also done on these sorted cells, with positively sorted cells forming the highest and negatively sorted cells the lowest percent of conjugates. Our data therefore indicates the important role of lysosome rich cells in the LAK cell population in the murine system.This work was supported by NIH grants R01 CA42962 and K04 CA0122, and by intramural funds from the Norris Cancer Center