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

OBJECTIVE: To explore the clinical features, lysosomal enzymatic [acid α-glucosidase (GAA)] activities and genetic variants in a child with late-onset Pompe disease (LOPD). METHODS: Clinical data of a child who had presented at the Genetic Counseling Clinic of West China Second University Hospital in August 2020 was retrospectively analyzed. Blood samples were collected from the patient and her parents for the isolation of leukocytes and lymphocytes as well as DNA extraction. The activity of lysosomal enzyme GAA in leukocytes and lymphocytes was analyzed with or without addition of inhibitor of GAA isozyme. Potential variants in genes associated with neuromuscular disorders were analyzed, in addition with conservation of the variant sites and protein structure. The remaining samples from 20 individuals undergoing peripheral blood lymphocyte chromosomal karyotyping were mixed and used as the normal reference for the enzymatic activities. RESULTS: The child, a 9-year-old female, had featured delayed language and motor development from 2 years and 11 months. Physical examination revealed unstable walking, difficulty in going upstairs and obvious scoliosis. Her serum creatine kinase was significantly increased, along with abnormal electromyography, whilst no abnormality was found by cardiac ultrasound. Genetic testing revealed that she has harbored compound heterozygous variants of the GAA gene, namely c.1996dupG (p.A666Gfs*71) (maternal) and c.701C>T (p.T234M) (paternal). Based on the guidelines from the American College of Medical Genetics and Genomics, the c.1996dupG (p.A666Gfs*71) was rated as pathogenic (PVS1+PM2_Supporting+PM3), whilst the c.701C>T (p.T234M) was rated as likely pathogenic (PM1+PM2_Supporting+PM3+PM5+PP3). The GAA in the leukocytes from the patient, her father and mother were respectively 76.1%, 91.3% and 95.6% of the normal value without the inhibitor, and 70.8%, 112.9% and 128.2% of the normal value with the inhibitor, whilst the activity of GAA in their leukocytes had decreased by 6 ~ 9 times after adding the inhibitor. GAA in lymphocytes of the patient, her father and mother were 68.3%, 59.0% and 59.5% of the normal value without the inhibitor, and 41.0%, 89.5% and 57.7% of the normal value with the inhibitor, the activity of GAA in lymphocytes has decreased by 2 ~ 5 times after adding the inhibitor. CONCLUSION The child was diagnosed with LOPD due to the c.1996dupG and c.701C>T compound heterozygous variants of the GAA gene. The residual activity of GAA among LOPD patients can range widely and the changes may be atypical. The diagnosis of LOPD should not be based solely on the results of enzymatic activity but combined clinical manifestation, genetic testing and measurement of enzymatic activity.
Humans ; Child ; Male ; Female ; Glycogen Storage Disease Type II/pathology* ; Retrospective Studies ; alpha-Glucosidases/genetics* ; Mothers ; Lysosomes/pathology* ; Mutation

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A, resulting in the accumulation of glycosphingolipids within the lysosomes of various cell types. It has a wide spectrum of clinical phenotypes, and renal failure is a serious complication. Fabry disease is confirmed either by measurement of α-galactosidase A activity or by genetic testing for GLA mutations. Renal biopsy findings on light microscopy, specifically enlarged podocytes with foamy cytoplasm, and osmiophilic inclusion bodies in the cytoplasm in all types of renal cells on electron microscopy, are characteristic of this disease. The predominant differential diagnosis is iatrogenic phospholipidosis in association with certain drugs that can cause cellular injuries indistinguishable from Fabry disease. Here, we report the case of a 10-year-old boy with microscopic hematuria who underwent a renal biopsy that showed morphological findings consistent with Fabry disease, although the patient had neither a GLA mutation nor a history of drug consumption. Six years later, spontaneous regression of this renal pathology was observed in a second renal biopsy examination.
Biopsy* ; Child* ; Cytoplasm ; Diagnosis, Differential ; Fabry Disease* ; Genetic Testing ; Glycosphingolipids ; Hematuria* ; Humans ; Inclusion Bodies ; Lysosomes ; Male* ; Microscopy ; Microscopy, Electron ; Pathology ; Phenotype ; Podocytes ; Renal Insufficiency

OBJECTIVETo study lysosomes involvement in the degradation of ricin A chain.

METHODSA lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain (rRTA) and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B. The cytotoxicity of recombinant proteins was measured by the MTT method.

RESULTSRecombinant RTA-KFERQ was 49.87%, 54.18% and 88.68% less cytotoxic than RTA itself on the three cell lines HEPG2, Hela and A549, respectively.

CONCLUSIONLysosomes can degrade, but not completely inactivate RTA in different cells, suggesting cells may have other degradation pathways for RTA.

Chromatography, Affinity ; Escherichia coli ; genetics ; metabolism ; HeLa Cells ; Humans ; Lung Neoplasms ; pathology ; Lysosomes ; metabolism ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism ; Ricin ; genetics ; metabolism ; Tumor Cells, Cultured

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 study the expression of cathepsin-B and -D in different time point after traumatic brain injury. METHODS: Traumatic brain injury (TBI) model was established on rats, cathepsin-B and cathepsin-D immunofluorescence staining and confocal microscope analysis were performed. Positive cells were counted by confocal microscope and image analysis techniques were used to determine the morphological changes in each group. RESULTS: Immunofluorescence staining results showed that cathepsin-B was activated 1 hour after TBI while cathepsin-D was not activated until 12hour after TBI. Both of them got to their peak during 4 to 8days, and kept a high level of activating 32days after TBI. Cathepsin-B and -D positive cells did not merge with caspase-3 positive cells until 6 h after TBI. CONCLUSION Cathepsin-B and -D could be the diagnostic markers of TBI and can estimating time course of lateral TBI. They blocked caspase-3 activation at the beginning period after TBI and started to promote cell death with caspase-3 6 h after TBI.
Animals ; Brain/pathology* ; Brain Injuries/pathology* ; Caspase 3/metabolism* ; Cathepsin B/metabolism* ; Cathepsin D/metabolism* ; Disease Models, Animal ; Forensic Pathology ; Hippocampus/pathology* ; Immunohistochemistry ; Lysosomes ; Male ; Neurons/metabolism* ; Rats ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVEThe aim of this study was to investigate the mechanisms of C. minima in the treatment of allergic rhinitis.

METHODAn allergic rhinitis animal model induced by ragweed pollen was established. After treatment with an active extract of C. minima, histopathological changes in the nasal mucosa of guinea pig were observed by transmission electron microscope.

RESULTIn the allergeic rhinitis model group, there appear a large number of lysosomes in the nasal epithelium with organelles vacuolated and nucleus deformed. Cells in the proper lamina of connective tissue were disarranged with organelles damaged, and there was also infiltration of eosinophils and mast cells in the connective tissue. However, in the treatment group receiving C. minima extract, the pathological changes mentioned above were significantly decreased.

CONCLUSIONC. minima is effective in treating allergic rhinitis.

Animals ; Asteraceae ; chemistry ; Epithelium ; ultrastructure ; Female ; Guinea Pigs ; Lysosomes ; drug effects ; Male ; Mitochondrial Swelling ; drug effects ; Nasal Mucosa ; pathology ; ultrastructure ; Oils, Volatile ; isolation & purification ; pharmacology ; Phytotherapy ; Plants, Medicinal ; chemistry ; Rhinitis, Allergic, Seasonal ; drug therapy ; pathology

Lysosomes are degradation and signaling centers within the cell, and their dysfunction impairs a wide variety of cellular processes. To understand the cellular effect of lysosome damage, we screened natural small-molecule compounds that induce lysosomal abnormality using Caenorhabditis elegans (C. elegans) as a model system. A group of vobasinyl-ibogan type bisindole alkaloids (ervachinines A-D) were identified that caused lysosome enlargement in C. elegans macrophage-like cells. Intriguingly, these compounds triggered cell death in the germ line independently of the canonical apoptosis pathway. In mammalian cells, ervachinines A-D induced lysosomal enlargement and damage, leading to leakage of cathepsin proteases, inhibition of autophagosome degradation and necrotic cell death. Further analysis revealed that this ervachinine-induced lysosome damage and lysosomal cell death depended on STAT3 signaling, but not RIP1 or RIP3 signaling. These findings suggest that lysosome-damaging compounds are promising reagents for dissecting signaling mechanisms underlying lysosome homeostasis and lysosome-related human disorders.
Alkaloids ; pharmacology ; Animals ; Caenorhabditis elegans ; cytology ; drug effects ; metabolism ; Cell Death ; drug effects ; Cell Survival ; drug effects ; HeLa Cells ; Humans ; Lysosomes ; drug effects ; pathology ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects

TRPML1 channel is a non-selective group-2 transient receptor potential (TRP) channel with Ca permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV). In the present study, we determined the cryo-electron microscopy (cryo-EM) structures of Mus musculus TRPML1 (mTRPML1) in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD) may sense the luminal/extracellular stimuli and undergo a "move upward" motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca, and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.
Animals ; Calcium ; metabolism ; Cryoelectron Microscopy ; Endocytosis ; Endosomes ; metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; metabolism ; Mice ; Models, Biological ; Mucolipidoses ; genetics ; metabolism ; pathology ; Nanostructures ; chemistry ; ultrastructure ; Phosphatidylinositols ; metabolism ; Transgenes ; Transient Receptor Potential Channels ; chemistry ; genetics ; metabolism

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.
Animals ; Autophagy ; physiology ; Beclin-1 ; metabolism ; Brain ; metabolism ; pathology ; Brain Ischemia ; metabolism ; pathology ; therapy ; Cystatin C ; genetics ; metabolism ; Disease Models, Animal ; Gene Expression ; Gene Knockdown Techniques ; Hyperbaric Oxygenation ; Lysosomes ; metabolism ; pathology ; Male ; Microtubule-Associated Proteins ; metabolism ; Neurons ; metabolism ; pathology ; Neuroprotection ; physiology ; Oxygen ; therapeutic use ; Random Allocation ; Rats, Sprague-Dawley ; Rats, Transgenic ; Reperfusion Injury ; metabolism ; pathology ; therapy