OBJECTIVETo observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.

METHODSThe localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.

RESULTSH(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.

CONCLUSIONThis finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.

Animals ; Cell Membrane ; enzymology ; Hepatocytes ; cytology ; enzymology ; ultrastructure ; Histocytochemistry ; methods ; Kidney ; cytology ; enzymology ; ultrastructure ; Lysosomes ; enzymology ; Male ; Organelles ; enzymology ; Rats ; Rats, Wistar ; Vacuolar Proton-Translocating ATPases ; metabolism

Apoptosis ; Calcineurin ; metabolism ; Chymotrypsin ; metabolism ; Humans ; Lysosomes ; enzymology ; Mitochondria ; metabolism ; pathology ; Mitochondrial Dynamics ; Neuroblastoma ; metabolism ; pathology

There are three different types of cell death, including apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). Ischemic neuronal death influences stroke development and progression. Lysosomes are important organelles having an acidic milieu to maintain cellular metabolism by degrading unneeded extra- and intracellular substances. Lysosomal enzymes, including cathepsins and some lipid hydrolases, when secreted following rupture of the lysosomal membrane, can be very harmful to their environment, which results in pathological destruction of cellular structures. Since lysosomes contain catalytic enzymes for degrading proteins, carbohydrates and lipids, it seems natural that they should participate in cellular death and dismantling. In this review, we discuss the recent developments in ischemic neuronal death, and present the possible molecular mechanisms that the lysosomal enzymes participate in the three different types of cell death in ischemic brain damage. Moreover, the research related to the selective cathepsin inhibitors may provide a novel therapeutic target for treating stroke and promoting recovery.
Animals ; Apoptosis ; Autophagy ; Brain Infarction ; enzymology ; physiopathology ; Brain Ischemia ; enzymology ; physiopathology ; Cathepsins ; metabolism ; Humans ; Lysosomes ; metabolism ; Necrosis ; physiopathology ; Nerve Degeneration ; enzymology ; physiopathology ; Peptide Hydrolases ; metabolism

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-beta, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.
Amino Acid Motifs ; Amino Acid Sequence ; Amino Acids/chemistry ; Cell Nucleus/*enzymology ; Endosomes/enzymology ; HEK293 Cells ; Humans ; Hydrophobic and Hydrophilic Interactions ; Lysosomes/enzymology ; Phagosomes/enzymology ; Phospholipase D/chemistry/*metabolism ; Protein Interaction Domains and Motifs ; Protein Transport ; Transport Vesicles/*enzymology

OBJECTIVETo explore the clinical characteristics of Wolman disease and diagnostic methods using enzymatic and molecular analysis.

METHODLysosomal acid lipase activity was measured using 4-methylumbelliferyl oleate in the leukocytes of an infant suspected of Wolman disease and LIPA gene mutational analysis was performed by PCR and direct sequencing in the proband and his parents. After the diagnosis was confirmed, the clinical, biochemical, radiological and histopathological findings in this case of Wolman disease were retrospectively reviewed.

RESULTThe sixteen-day-old boy was failing to thrive with progressive vomiting, abdominal distention and hepatosplenomegaly. Abdominal X-ray revealed adrenal calcifications which were confirmed on abdominal CT scan. Xanthomatosis were observed on enlarged liver, spleen and lymph nodes during abdominal surgery. Liver and lymph node biopsy showed foamy histiocytes. The lysosomal acid lipase activity in leukocytes was 3.5 nmol/(mg·h) [control 35.5 - 105.8 nmol/(mg·h)]. Serum chitotriosidase activity was 315.8 nmol/(ml·h) [control 0 - 53 nmol/(ml·h)]. The patient was homozygote for a novel insert mutation allele c.318 ins T, p. Phe106fsX4 in exon 4 on LIPA gene. His both parents were carriers of the mutation.

CONCLUSIONThe clinical features of Wolman disease include early onset of vomiting, abdominal distention, growth failure, hepatosplenomegaly and bilateral adrenal calcification after birth. A plain abdominal X-ray film should be taken to check for the typical pattern of adrenal calcification in suspected cases of Wolman disease. The enzymatic and molecular analyses of lysosomal acid lipase can confirm the diagnosis of Wolman disease.

Adrenal Gland Diseases ; etiology ; pathology ; Exons ; Humans ; Infant, Newborn ; Leukocytes ; enzymology ; Lipase ; blood ; genetics ; Liver ; pathology ; Lysosomes ; enzymology ; genetics ; Male ; Mutation ; Polymerase Chain Reaction ; Splenomegaly ; pathology ; Sterol Esterase ; genetics ; Tomography, X-Ray Computed ; Wolman Disease ; diagnosis ; enzymology ; genetics ; pathology

OBJECTIVE: To determine the distribution and influence of lysosomal neuraminidase (Neul), protective protein/cathepsin A (PPCA) and beta-galactosidase (beta-gal) in the inner ear of the mouse, and to observe their auditory alterations in enzyme deficiency. METHODS: Six wild type (2 months postnatal) (Neu1+/+, PPCA+/+ and beta-gal+/+) mice were used, and Neu1, PPCA and beta-gal homozygous (Neu1-/-, PPCA-/- and beta-gal-/-) mice at the same age used as control in this experiment. The auditory thresholds were examined through the auditory brainstem responses (ABR) to click, which tone pips were 8, 16, and 32 kHz. The mice were intracardically perfused with 4% paraformaldehyde. The bulla were further fixed in 4% paraformaldehyde, processed and sectioned with paraffin embedded method. Immunohistochemistry was used to determine the cellular localizations of Neu1, PP-CA, and beta gal in the inner ear. RESULTS: There was a similar distributive pattern of Neu1, PPCA and betagal in the inner ear. Neu1 intense staining was observed in the cochlear spiral ganglion cells, spiral limbus, spiral ligament, vestibular ganglion cells, cristae, maculae hair cells, and weak staining in inner hair cells, outer hair cells, supplying cells of the organ of Corti and stria vascularis. The intense staining of PPCA and beta-gal were observed in the spiral ganglion and vestibular ganglion cells, and weak staining in the spiral limbus, spiral ligament, stria vascularis and organ of Corti. The inner ear exhibited no staining when Neul, PPCA and beta-gal were deficient, respectively. A positive staining of PPCA and beta-gal was presented in Neu1-/- mice, and as well as Neu1 and PPCA in beta-gal-/- mice. However, the staining of Neu1 was not presented, and only very weak staining of beta-gal in PPCA-/- mice. The auditory thresholds of Neul, PPCA, and beta-gal mice were elevated for 60-69 dB, 40-48 dB, and 7-10 dB above those of wildtype littermates, respectively. CONCLUSION Neu1 PPCA and beta-gal are distributed in the inner ear of mouse, and the three enzymes also form a lysosomal multi-enzyme complex in the inner ear. The respective enzyme deficiencies can induce the hearing the loss of different levels.
Animals ; Auditory Threshold ; Cathepsin A ; genetics ; metabolism ; Ear, Inner ; enzymology ; Evoked Potentials, Auditory, Brain Stem ; physiology ; Hearing Loss, Sensorineural ; enzymology ; genetics ; Lysosomes ; enzymology ; Mice ; Mice, Knockout ; Neuraminidase ; genetics ; metabolism ; beta-Galactosidase ; genetics ; metabolism

OBJECTIVETo study the effect of imbalance between lysosomal enzymes and their inhibitors in blood on disturbance of the local and whole body after trauma.

METHODSThe dynamic changes of lysosomal enzymes and proteinase inhibitors were studied in 12 pigs with femoral comminuted fractures in both hind limbs caused by high velocity missiles. Four normal pigs served as controls.

RESULTSAfter injury, the activity of Cathepsin D in arterial plasma increased gradually and reached the highest level at 8 hours, acid phosphatase in serum began to increase at 12 hours and the value of serum elastase did not change significantly. The level of alpha1-antitrypsin, a proteinase inhibitor in plasma, decreased significantly in the early stage after injury [73.5%+/-6.4% and 81.0%+/-5.1% of the baseline value (1.67 micromol x ml(-1) x min(-1)+/- 0.29 micromol x ml(-1) x min(-1)) at l and 2 hours after injury, respectively, P<0.05], then increased gradually and was higher than the baseline value at 12 hours after injury.

CONCLUSIONSImbalance between lysosomal enzymes and proteinase inhibitors occurs soon after injury, which might result in continuous tissue damage and play an important role in the disturbance of general reaction after injury.

Acid Phosphatase ; blood ; Animals ; Cathepsin D ; blood ; Endopeptidases ; blood ; Female ; Lysosomes ; enzymology ; Male ; Pancreatic Elastase ; blood ; Swine ; Wounds, Gunshot ; blood ; alpha 1-Antitrypsin ; analysis

In this study, we amplified human lysosomal acid beta-glucosidase (GlcCerase) gene by RT-PCR from human placenta, and analyzed the sequence of the PCR product cloned in pMD-19T. The gene identity was 99% comparable to that of the reported human GlcCerase cDNA sequence in GenBank. The GlcCerase gene digested with Xho I was subcloned into eukaryotic express vector pEGFP-C1 to generate recombinant expression vector pEGFP-GlcCerase. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pEGFP-GlcCerase into COS7 cells by liposome. GlcCerase mRNA was expressed and the activity of GlcCerase was also detected in COS7 cells. This study would lay a foundation for the function of GlcCerase and its production by transgenic bioreactor.
Animals ; COS Cells ; Cercopithecus aethiops ; Cloning, Molecular ; Genetic Vectors ; genetics ; Glucosylceramidase ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; Humans ; Lysosomes ; enzymology ; Recombination, Genetic ; Transfection

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway and is encoded by a 3.0 kb cDNA. A 2.3 kb cDNA from a minor species of HeLa cell mRNA was discovered by RT-PCR cloning. Southern blotting and PCR analysis of the HeLa cell genomic DNA showed that the 2.3 kb message was encoded by the lysosomal alpha-mannosidase gene. Sequence comparison of the cDNA with the corresponding genomic DNA indicated that the 2.3 kb message was generated by an unusual intra-exonic joining event.
*Alternative Splicing ; Base Sequence ; DNA, Complementary/genetics ; Exons ; Hela Cells ; Human ; Lysosomes/*enzymology ; Mannosidases/*genetics ; Molecular Sequence Data ; RNA, Messenger/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Support, U.S. Gov't, P.H.S.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is a major exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease, alpha-mannosidosis, which has been described in humans, cattle, domestic cats and guinea pigs. Recently, great progress has been made in studying the enzyme and its deficiency. This includes cloning of the gene encoding the enzyme, characterization of mutations related to the disease, establishment of valuable animal models, and encouraging results from bone marrow transplantation experiments.
Animal ; Cats ; Cattle ; Cloning, Molecular ; Disease Models, Animal ; Guinea Pigs ; Human ; Lysosomes/*enzymology ; Mannosidases/*deficiency/*genetics/metabolism ; Mannosidosis/diagnosis/*etiology/*therapy ; Mutation ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic