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Reproductive Biology and Endocrinology... Dec 2009Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper...
BACKGROUND
Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer.
METHODS
First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+)-free medium. The potential of the most promising treatments (with or without subsequent incubation in the presence of cycloheximide and cytochalasin B) to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats.
RESULTS
All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7%) and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%); incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos (a total of 220) were transferred into 19 recipient females, 4 animals became pregnant. All of the fetuses developed from oocytes activated by electroporation followed by cycloheximide and cytochalasin B incubation; no fetal development was detected as a result of thimerosal/DTT activation. Although heartbeats were detected in two of the cloned fetuses, no term development occurred.
CONCLUSION
Electroporation proved to be the most effective method for the activation of cat oocytes reconstructed by nuclear transfer. The combined thimerosal/DTT treatment followed by cycloheximide and cytochalasin B incubation triggered development effectively to the blastocyst stage; whether it is a viable option to stimulate term development of cloned cat embryos needs further investigations.
Topics: Animals; Calcium; Cat Diseases; Cats; Cycloheximide; Cytochalasin B; Disease Models, Animal; Electroporation; Embryo Culture Techniques; Embryonic Development; Female; Fibroblasts; Mucopolysaccharidosis I; Nuclear Transfer Techniques; Oocytes; Parthenogenesis; Pregnancy; Preservatives, Pharmaceutical; Protein Synthesis Inhibitors; Stimulation, Chemical; Thimerosal; alpha-Mannosidosis
PubMed: 20003339
DOI: 10.1186/1477-7827-7-148 -
Molecular and Cellular Biology Jan 2010Alpha-mannosidosis is caused by the genetic defect of the lysosomal alpha-d-mannosidase (LAMAN), which is involved in the breakdown of free alpha-linked...
Alpha-mannosidosis is caused by the genetic defect of the lysosomal alpha-d-mannosidase (LAMAN), which is involved in the breakdown of free alpha-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with alpha-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with alpha-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human alpha-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparagine-linked oligosaccharides on native lysosomal proteins.
Topics: Animals; Cathepsin B; Enzyme Replacement Therapy; Glycosylation; Humans; Liver; Lysosomes; Mice; Mice, Inbred C57BL; Oligosaccharides; Proteins; Recombinant Proteins; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 19884343
DOI: 10.1128/MCB.01143-09 -
NMR in Biomedicine Jan 2010A genetic deficiency of lysosomal alpha-mannosidase causes the lysosomal storage disease alpha-mannosidosis (AMD), in which oligosaccharide accumulation occurs in...
A genetic deficiency of lysosomal alpha-mannosidase causes the lysosomal storage disease alpha-mannosidosis (AMD), in which oligosaccharide accumulation occurs in neurons and glia. The purpose of this study was to evaluate the role of magnetic resonance spectroscopy (MRS) in detecting the oligosaccharide accumulation in AMD. Five cats with AMD and eight age-matched normal cats underwent in vivo MRS studies with a single voxel short echo time (20 ms) STEAM spectroscopy sequence on a 4.7T magnet. Two voxels were studied in each cat, from the cerebellar vermis and the occipital cortex. Metabolites of brain samples from these regions were extracted with perchloric acid and analyzed by high resolution NMR spectroscopy. A significantly elevated unresolved resonance signal between 3.4 and 4. ppm was observed in the cerebellar vermis and occipital cortex of all AMD cats, which was absent in normal cats. This resonance was shown to be from carbohydrate moieties by high resolution NMR of tissue extracts. Resonances from the Glc-NAc group (1.8-2.2 ppm) along with anomeric proton signals (4.6-5.4 ppm) from undigested oligosaccharides were also observed in the extract spectra from AMD cats. This MRS spectral pattern may be a useful biomarker for AMD diagnosis as well as for assessing responses to therapy.
Topics: Animals; Biomarkers; Cats; Cerebellum; Humans; Magnetic Resonance Spectroscopy; Occipital Lobe; Oligosaccharides; alpha-Mannosidosis
PubMed: 19743435
DOI: 10.1002/nbm.1430 -
Transplantation Aug 2009Lysosomal storage diseases are devastating illnesses, in large part because of their neurologic consequences. Because significant morbidity occurs prenatally, in utero...
BACKGROUND
Lysosomal storage diseases are devastating illnesses, in large part because of their neurologic consequences. Because significant morbidity occurs prenatally, in utero (IU) therapy is an attractive therapeutic approach.
METHODS
We studied the feasibility and efficacy of IU injections of monocytic cells (derived from normal marrow) in feline alpha-mannosidosis. Heterozygous cats were interbred to produce affected (homozygous) and control (heterozygous and wild-type) offspring. Thirty-seven pregnancies were studied in which fetuses were transplanted intraperitoneally (1x10 cells/kg recipient) at gestational days 27 to 33 and then each week for 2 weeks (term=63 days). After birth, affected kittens were evaluated clinically and pathologically, tissue alpha-mannosidase levels were assayed, and in many studies, the numbers of alpha-mannosidase-containing cells were enumerated. When male donor cells were transplanted into female recipients, engraftment was also quantified using polymerase chain reaction to amplify a Y chromosome-specific sequence.
RESULTS
We establish methods to transplant cats intraperitoneally while IU using ultrasound guidance, thus, describing a new large animal model for prenatal therapy. We show that the donor monocytic cells engraft and persist (for up to 125 days) in the brain, liver, and spleen, albeit at levels below those needed to alter the clinical or pathological progression of the alpha-mannosidosis.
CONCLUSIONS
This is the first study of monocyte transplantation in a large animal model of a lysosomal storage disorder and demonstrates its feasibility, safety, and promise. Delivering cells IU may be a useful strategy to prevent morbidities before a definitive therapy, such as hematopoietic stem-cell transplantation, can be administered after birth.
Topics: Animals; Animals, Newborn; Bone Marrow Transplantation; Brain; Cats; Cell Survival; Cells, Cultured; Disease Models, Animal; Feasibility Studies; Female; Gestational Age; Injections, Intraperitoneal; Liver; Male; Monocytes; Pregnancy; Spleen; Time Factors; Ultrasonography, Interventional; Uterus; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 19667933
DOI: 10.1097/TP.0b013e3181b0d264 -
Orphanet Journal of Rare Diseases Jul 2008Alpha-mannosidosis is an inherited lysosomal storage disorder characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual... (Review)
Review
Alpha-mannosidosis is an inherited lysosomal storage disorder characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual disability. It occurs in approximately 1 of 500,000 live births. The children are often born apparently normal, and their condition worsens progressively. Some children are born with ankle equinus or develop hydrocephalus in the first year of life. Main features are immune deficiency (manifested by recurrent infections, especially in the first decade of life), skeletal abnormalities (mild-to-moderate dysostosis multiplex, scoliosis and deformation of the sternum), hearing impairment (moderate-to-severe sensorineural hearing loss), gradual impairment of mental functions and speech, and often, periods of psychosis. Associated motor function disturbances include muscular weakness, joint abnormalities and ataxia. The facial trait include large head with prominent forehead, rounded eyebrows, flattened nasal bridge, macroglossia, widely spaced teeth, and prognathism. Slight strabismus is common. The clinical variability is significant, representing a continuum in severity. The disorder is caused by lysosomal alpha-mannosidase deficiency. Alpha-mannosidosis is inherited in an autosomal recessive fashion and is caused by mutations in the MAN2B1 gene located on chromosome 19 (19 p13.2-q12). Diagnosis is made by measuring acid alpha-mannosidase activity in leukocytes or other nucleated cells and can be confirmed by genetic testing. Elevated urinary secretion of mannose-rich oligosaccharides is suggestive, but not diagnostic. Differential diagnoses are mainly the other lysosomal storage diseases like the mucopolysaccharidoses. Genetic counseling should be given to explain the nature of the disease and to detect carriers. Antenatal diagnosis is possible, based on both biochemical and genetic methods. The management should be pro-active, preventing complications and treating manifestations. Infections must be treated frequently. Otolaryngological treatment of fluid in the middle ear is often required and use of hearing aids is invariably required. Early educational intervention for development of social skills is needed and physiotherapy is important to improve bodily function. Orthopedic surgery may be necessary. The long-term prognosis is poor. There is an insidiously slow progression of neuromuscular and skeletal deterioration over several decades, making most patients wheel-chair dependent. No patients manage to be completely socially independent. Many patients are over 50 years of age.
Topics: Diagnosis, Differential; Genetic Counseling; Humans; Models, Molecular; Mutation; Prognosis; Protein Conformation; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 18651971
DOI: 10.1186/1750-1172-3-21 -
Anales de Pediatria (Barcelona, Spain :... Mar 2008
Topics: Child, Preschool; Diagnosis, Differential; Health Status; Humans; Male; Phenotype; Severity of Illness Index; alpha-Mannosidosis
PubMed: 18358145
DOI: 10.1157/13116714 -
PLoS Biology Feb 2008A lysosomal storage disease (LSD) results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to...
A lysosomal storage disease (LSD) results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to pathology. In many but not all LSDs, the clinically most important mutations compromise the cellular folding of the enzyme, subjecting it to endoplasmic reticulum-associated degradation instead of proper folding and lysosomal trafficking. A small molecule that restores partial mutant enzyme folding, trafficking, and activity would be highly desirable, particularly if one molecule could ameliorate multiple distinct LSDs by virtue of its mechanism of action. Inhibition of L-type Ca2+ channels, using either diltiazem or verapamil-both US Food and Drug Administration-approved hypertension drugs-partially restores N370S and L444P glucocerebrosidase homeostasis in Gaucher patient-derived fibroblasts; the latter mutation is associated with refractory neuropathic disease. Diltiazem structure-activity studies suggest that it is its Ca2+ channel blocker activity that enhances the capacity of the endoplasmic reticulum to fold misfolding-prone proteins, likely by modest up-regulation of a subset of molecular chaperones, including BiP and Hsp40. Importantly, diltiazem and verapamil also partially restore mutant enzyme homeostasis in two other distinct LSDs involving enzymes essential for glycoprotein and heparan sulfate degradation, namely alpha-mannosidosis and type IIIA mucopolysaccharidosis, respectively. Manipulation of calcium homeostasis may represent a general strategy to restore protein homeostasis in multiple LSDs. However, further efforts are required to demonstrate clinical utility and safety.
Topics: Animals; Calcium; Cell Line; Homeostasis; Lysosomal Storage Diseases; Mutation
PubMed: 18254660
DOI: 10.1371/journal.pbio.0060026 -
Developmental Medicine and Child... Nov 2007Alpha-mannosidosis (AMS) is an autosomal recessive lysosomal storage disorder which results from a deficiency of lysosomal alpha-mannosidase [corrected] activity and...
Alpha-mannosidosis (AMS) is an autosomal recessive lysosomal storage disorder which results from a deficiency of lysosomal alpha-mannosidase [corrected] activity and displays a wide range of clinical phenotypes. Patients have traditionally been divided into type I, a more severe form that presents in infancy, and type II, a milder form that typically presents in later childhood. We describe three Hispanic males who presented in infancy with relatively mild forms of AMS. They were aged between 6 and 24 years at their last assessment. Homozygous mutations in the MAN2B1 gene were found in all three patients, one of which is a newly reported mutation. Two of the patients were brothers who were homozygous for the same MAN2B1 mutation. Despite being homozygous for the same mutation, the older brother had more severe developmental delay, hearing loss, and growth retardation. This report illustrates the difficulty in determining a strict genotype-phenotype correlation in AMS, and supports screening for oligosaccharides in children with neurodevelopmental delay with mild phenotypic signs and symptoms.
Topics: Adult; Age Factors; Child; Disease Progression; Early Diagnosis; Hispanic or Latino; Humans; Male; Muscle Hypotonia; Phenotype; alpha-Mannosidosis
PubMed: 17979865
DOI: 10.1111/j.1469-8749.2007.00854.x -
AJNR. American Journal of Neuroradiology Feb 2008Methods to locate and identify brain pathology are critical for monitoring disease progression and for evaluating the efficacy of therapeutic intervention. The purpose...
BACKGROUND AND PURPOSE
Methods to locate and identify brain pathology are critical for monitoring disease progression and for evaluating the efficacy of therapeutic intervention. The purpose of this study was to detect cell swelling, abnormal myelin, and astrogliosis in the feline model of the lysosomal storage disease alpha-mannosidosis (AMD) by using diffusion and T2 mapping.
MATERIALS AND METHODS
Average apparent diffusion coefficient (ADC(av)) and T2 were measured by imaging the brains of five 16-week-old cats with feline AMD on a 4.7T magnet. ADC(av) and T2 data from affected cats were compared with data from age-matched normal cats. Brains were collected from both affected and normal cats following imaging, and histology was compared with quantitative imaging data.
RESULTS
Gray matter from AMD cats demonstrated a 13%-15% decrease in ADC(av) compared with that in normal cats. White matter from AMD cats exhibited an 11%-16% decrease in ADC(av) and a 5%-12% increase in T2 values compared with those in normal control cats. Histologic evidence of neuronal and glial swelling, abnormal myelin, and astrogliosis was consistent with changes in ADC(av) and T2.
CONCLUSION
ADC(av) and T2 data can be used to quantify differences in the gray and white matter in the feline AMD brain and may serve as surrogate markers of neuronal swelling, abnormal myelin, and astrogliosis associated with this disease. These studies may be helpful in assessing the efficacy of experimental therapies for central nervous system disease associated with lysosomal storage diseases.
Topics: Animals; Brain; Cat Diseases; Cats; Demyelinating Diseases; Diffusion Magnetic Resonance Imaging; Image Interpretation, Computer-Assisted; Nerve Fibers, Myelinated; Neurons; Reproducibility of Results; Sensitivity and Specificity; alpha-Mannosidosis
PubMed: 17974615
DOI: 10.3174/ajnr.A0791 -
Cell Aug 2007Autoimmune disease is typically defined as an aberrant response of lymphocytes to self antigens that ultimately leads to tissue damage. Reporting in Immunity, Green et...
Autoimmune disease is typically defined as an aberrant response of lymphocytes to self antigens that ultimately leads to tissue damage. Reporting in Immunity, Green et al. (2007) now show that mice lacking alpha-mannosidase II develop an autoimmune disease similar to lupus. Remarkably, this illness is precipitated by an innate immune response to altered self glycans that mimic molecular patterns found on pathogens.
Topics: Aging; Animals; Autoimmune Diseases; Dose-Response Relationship, Drug; Homeodomain Proteins; Immunity, Innate; Immunoglobulins; Lupus Erythematosus, Systemic; Mannose-Binding Protein-Associated Serine Proteases; Mice; Mice, Knockout; Models, Immunological; alpha-Mannosidosis
PubMed: 17719535
DOI: 10.1016/j.cell.2007.08.009