A substantial lack of information is recognized on the features underlying the variable susceptib... more A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Aβ1-40, Aβ1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca2+ and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca2+-ATPase equipment and a higher total an...
The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experi... more The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experimental evidence suggests that reducing circulating and brain cholesterol protects against AD, however recent data indicate that low membrane cholesterol results in neurode-generation and that the cholesterol synthesis catalyst seladin-1 is down-regulated in AD-affected brain regions. We previously reported a significant correlation between resistance to amyloid toxicity and content of membrane cholesterol in differing cultured cell types. Here we provide evidence that Aβ42 pre-fibrillar aggregates accumulate more slowly and in reduced amount at the plasma membrane of human SH-SY5Y neuroblastoma cells overexpressing seladin-1 or treated with PEG-cholesterol than at the membrane of control cells. The accumulation was significantly increased in cholesterol-depleted cells following treatment with the specific seladin-1 inhibitor 5,22E-cholestadien-3-ol or with methyl-β-cyclodextrin. The resistance to amyloid toxicity and the early cytosolic Ca2+ rise following exposure to Aβ42 aggregates were increased and prevented, respectively, by increasing membrane cholesterol whereas the opposite effects were found in cholesterol-depleted cells. These results suggest that seladin-1-dependent cholesterol synthesis reduces membrane-aggregate interaction and cell damage associated to amyloid-induced imbalance of cytosolic Ca2+. Our findings extend recently reported data indicating that seladin-1 overexpression directly enhances the resistance to Aβ toxicity featuring seladin-1/DHCR 24 as a possible new susceptibility gene for sporadic AD.
A fast and sensitive method for the isolation and quantitation of cytoplasmic ubiquitin from brai... more A fast and sensitive method for the isolation and quantitation of cytoplasmic ubiquitin from brain by reversed-phase high-performance liquid chromatography is described. Cytosol from brain tissue was obtained by differential centrifugation and, after perchloric acid treatment, the sample was concentrated and ubiquitin was quantitatively isolated by means of a single chromatographic run. The amino acid composition, molecular weight, and primary structure of the pure protein were identified. The addition of monoiodinated 125I-ubiquitin to the sample as an internal standard indicated high native ubiquitin recovery. Statistical analysis carried out on different preparations and standardization of the chromatographic system indicated both the accuracy and the reproducibility of the method.
Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma... more Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma-glutamyl cysteine synthetase (gamma-GCS) were measured in lymphoblast lines from patients with familial and sporadic Alzheimer's disease (AD) and from age-matched controls. Lymphoblasts carrying presenilins (PS) and amyloid precursor protein (APP) genes mutations showed significantly decreased GSH content with respect to controls. Levels of GSSG and glutamic acid, as well as the activity of gamma-GCS were not significantly different in lymphoblasts carrying genes mutations as compared with control cells. These results indicate that even peripheral cells not involved in the neurodegenerative process of AD show altered GSH content when carrying PS and APP genes mutations. The provided data appear to be in accordance with the known alteration of GSH levels in central nervous system and strengthen the hypothesis of oxidative stress as an important, possibly crucial mechanism in the pathogenesis of AD.
The post-mortem stability of some brain enzymes was studied. Over the time period under examinati... more The post-mortem stability of some brain enzymes was studied. Over the time period under examination, the cytoplasmic enzymes investigated underwent a decisive decay, hexokinase being the most labile and acylphosphatase the most stable. On the other hand, structured activities such as Na+, K+-ATPase and Ca2+, Mg2+-ATPase showed an apparent transitory increase. The differences in post-mortem stability of soluble enzymes could be ascribed, at least in part, to their different susceptibility toward proteolytic activities, as suggested by the electrophoretic results.
ABSTRACT The data presented in this paper concern a kinetic study of the calcium uptake by sarcop... more ABSTRACT The data presented in this paper concern a kinetic study of the calcium uptake by sarcoplasmic reticulum vesicles and of the hydrolysis of the substrates which support the process. The results show that substrates which are different from ATP, acetylphosphate, and carbamylphosphate are able to support calcium transport. The technique used to follow the process allows us to detect continuously the changes in the concentration of the calcium present in the external medium. In our experimental conditions the calcium uptake supported by all the high energy substrates tested proceeds for several seconds at a constant rate, presumably corresponding to the “steady state” of the process; furthermore the calcium transport is clearly Ca2+ and Mg2+ dependent: the lowering of the Ca+ concentration in the medium from 10−4 to 10−5m causes a remarkable reduction of the V of the calcium transport and an apparent increase of the affinity of the sarcoplasmic reticulum vesicles for the acylphosphates; in the absence of Mg2+, none of the substrates is able to support the calcium uptake which increases in the presence of rising amounts of Mg2+ in the reaction medium. Furthermore, both the calcium transport and the substrate hydrolysis appear to follow the Michaelis-Menten kinetics in the presence of acylphosphates but not in the presence of ATP. The hydrolytic activity of sarcoplasmic reticulum vesicles on ATP and acylphosphates reveals a clear Mg2+ dependence; furthermore, in the absence of free Ca2+ and in the presence of 5 mm Mg2+, the high energy substrates tested reveal a different susceptibility to the hydrolitic attack by sarcoplasmic reticulum vesicles.
Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degen... more Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degeneration of motor neurons in the brain and spinal cord. Approximately 20% of the inherited autosomal dominant cases are due to mutations within the gene coding for Cu/Zn superoxide dismutase 1 (SOD1), a cytosolic homodimeric enzyme that catalyzes the dismutation of toxic superoxide anion. We investigated the presence of SOD1 gene mutations and activity alterations in two unrelated families of ALS patients from Elba, an island of central Italy. No mutation in SOD1 exon 1 to 5 and no activity alteration were observed in all members of the two analyzed ALS families (FALS). These data show an apparent heterogeneous distribution of ALS patients with SOD1 gene mutations among different populations and suggest that another genetic locus could be involved in the disease.
A new acylphosphatase from human erythrocytes was isolated by an original purification procedure.... more A new acylphosphatase from human erythrocytes was isolated by an original purification procedure. It is an isoenzyme of the well-characterized human skeletal muscle acylphosphatase. The erythrocyte enzyme shows hydrolytic activity on acyl phosphates with higher affinity than the muscle enzyme for some substrates and phosphorylated inhibitors. The sequence was determined by characterizing the peptides purified from tryptic, peptic, and Staphylococcus aureus V8 protease digests of the protein, and it was found to differ in 44% of the total positions as compared to the human muscle enzyme. About one-third of these differences are in the form of strictly conservative replacements. The protein consists of 98 amino acid residues; it has an acetylated NH2-terminus and does not contain cysteine: (sequence in text).
Biochemical and Biophysical Research Communications, 1997
Levels of free intracellular calcium have been measured on two cell lines of cultured human fibro... more Levels of free intracellular calcium have been measured on two cell lines of cultured human fibroblasts carrying the genetic lesions occurring in Duchenne and Becker dystrophies. Both cell lines elicited a markedly higher content of the cation (98 nM and 57 nM, respectively) than control fibroblasts (35 nM). Differences toward controls were statistically significant (p < 0.01). Dystrophic fibroblasts were also found to possess a significantly reduced amount by about 50% of muscular acylphosphatase isoenzyme as compared to normal cells. As acylphosphatase was demonstrated to be involved in the regulation of Ca2+-ATPase activity from different sources, a hypothesis was formulated that could explain the disruption of calcium homeostasis as an effect of the altered acylphosphatase activity.
A substantial lack of information is recognized on the features underlying the variable susceptib... more A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Aβ1-40, Aβ1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca2+ and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca2+-ATPase equipment and a higher total an...
The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experi... more The role of brain cholesterol in Alzheimer's disease (AD) is currently a matter of debate. Experimental evidence suggests that reducing circulating and brain cholesterol protects against AD, however recent data indicate that low membrane cholesterol results in neurode-generation and that the cholesterol synthesis catalyst seladin-1 is down-regulated in AD-affected brain regions. We previously reported a significant correlation between resistance to amyloid toxicity and content of membrane cholesterol in differing cultured cell types. Here we provide evidence that Aβ42 pre-fibrillar aggregates accumulate more slowly and in reduced amount at the plasma membrane of human SH-SY5Y neuroblastoma cells overexpressing seladin-1 or treated with PEG-cholesterol than at the membrane of control cells. The accumulation was significantly increased in cholesterol-depleted cells following treatment with the specific seladin-1 inhibitor 5,22E-cholestadien-3-ol or with methyl-β-cyclodextrin. The resistance to amyloid toxicity and the early cytosolic Ca2+ rise following exposure to Aβ42 aggregates were increased and prevented, respectively, by increasing membrane cholesterol whereas the opposite effects were found in cholesterol-depleted cells. These results suggest that seladin-1-dependent cholesterol synthesis reduces membrane-aggregate interaction and cell damage associated to amyloid-induced imbalance of cytosolic Ca2+. Our findings extend recently reported data indicating that seladin-1 overexpression directly enhances the resistance to Aβ toxicity featuring seladin-1/DHCR 24 as a possible new susceptibility gene for sporadic AD.
A fast and sensitive method for the isolation and quantitation of cytoplasmic ubiquitin from brai... more A fast and sensitive method for the isolation and quantitation of cytoplasmic ubiquitin from brain by reversed-phase high-performance liquid chromatography is described. Cytosol from brain tissue was obtained by differential centrifugation and, after perchloric acid treatment, the sample was concentrated and ubiquitin was quantitatively isolated by means of a single chromatographic run. The amino acid composition, molecular weight, and primary structure of the pure protein were identified. The addition of monoiodinated 125I-ubiquitin to the sample as an internal standard indicated high native ubiquitin recovery. Statistical analysis carried out on different preparations and standardization of the chromatographic system indicated both the accuracy and the reproducibility of the method.
Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma... more Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma-glutamyl cysteine synthetase (gamma-GCS) were measured in lymphoblast lines from patients with familial and sporadic Alzheimer's disease (AD) and from age-matched controls. Lymphoblasts carrying presenilins (PS) and amyloid precursor protein (APP) genes mutations showed significantly decreased GSH content with respect to controls. Levels of GSSG and glutamic acid, as well as the activity of gamma-GCS were not significantly different in lymphoblasts carrying genes mutations as compared with control cells. These results indicate that even peripheral cells not involved in the neurodegenerative process of AD show altered GSH content when carrying PS and APP genes mutations. The provided data appear to be in accordance with the known alteration of GSH levels in central nervous system and strengthen the hypothesis of oxidative stress as an important, possibly crucial mechanism in the pathogenesis of AD.
The post-mortem stability of some brain enzymes was studied. Over the time period under examinati... more The post-mortem stability of some brain enzymes was studied. Over the time period under examination, the cytoplasmic enzymes investigated underwent a decisive decay, hexokinase being the most labile and acylphosphatase the most stable. On the other hand, structured activities such as Na+, K+-ATPase and Ca2+, Mg2+-ATPase showed an apparent transitory increase. The differences in post-mortem stability of soluble enzymes could be ascribed, at least in part, to their different susceptibility toward proteolytic activities, as suggested by the electrophoretic results.
ABSTRACT The data presented in this paper concern a kinetic study of the calcium uptake by sarcop... more ABSTRACT The data presented in this paper concern a kinetic study of the calcium uptake by sarcoplasmic reticulum vesicles and of the hydrolysis of the substrates which support the process. The results show that substrates which are different from ATP, acetylphosphate, and carbamylphosphate are able to support calcium transport. The technique used to follow the process allows us to detect continuously the changes in the concentration of the calcium present in the external medium. In our experimental conditions the calcium uptake supported by all the high energy substrates tested proceeds for several seconds at a constant rate, presumably corresponding to the “steady state” of the process; furthermore the calcium transport is clearly Ca2+ and Mg2+ dependent: the lowering of the Ca+ concentration in the medium from 10−4 to 10−5m causes a remarkable reduction of the V of the calcium transport and an apparent increase of the affinity of the sarcoplasmic reticulum vesicles for the acylphosphates; in the absence of Mg2+, none of the substrates is able to support the calcium uptake which increases in the presence of rising amounts of Mg2+ in the reaction medium. Furthermore, both the calcium transport and the substrate hydrolysis appear to follow the Michaelis-Menten kinetics in the presence of acylphosphates but not in the presence of ATP. The hydrolytic activity of sarcoplasmic reticulum vesicles on ATP and acylphosphates reveals a clear Mg2+ dependence; furthermore, in the absence of free Ca2+ and in the presence of 5 mm Mg2+, the high energy substrates tested reveal a different susceptibility to the hydrolitic attack by sarcoplasmic reticulum vesicles.
Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degen... more Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degeneration of motor neurons in the brain and spinal cord. Approximately 20% of the inherited autosomal dominant cases are due to mutations within the gene coding for Cu/Zn superoxide dismutase 1 (SOD1), a cytosolic homodimeric enzyme that catalyzes the dismutation of toxic superoxide anion. We investigated the presence of SOD1 gene mutations and activity alterations in two unrelated families of ALS patients from Elba, an island of central Italy. No mutation in SOD1 exon 1 to 5 and no activity alteration were observed in all members of the two analyzed ALS families (FALS). These data show an apparent heterogeneous distribution of ALS patients with SOD1 gene mutations among different populations and suggest that another genetic locus could be involved in the disease.
A new acylphosphatase from human erythrocytes was isolated by an original purification procedure.... more A new acylphosphatase from human erythrocytes was isolated by an original purification procedure. It is an isoenzyme of the well-characterized human skeletal muscle acylphosphatase. The erythrocyte enzyme shows hydrolytic activity on acyl phosphates with higher affinity than the muscle enzyme for some substrates and phosphorylated inhibitors. The sequence was determined by characterizing the peptides purified from tryptic, peptic, and Staphylococcus aureus V8 protease digests of the protein, and it was found to differ in 44% of the total positions as compared to the human muscle enzyme. About one-third of these differences are in the form of strictly conservative replacements. The protein consists of 98 amino acid residues; it has an acetylated NH2-terminus and does not contain cysteine: (sequence in text).
Biochemical and Biophysical Research Communications, 1997
Levels of free intracellular calcium have been measured on two cell lines of cultured human fibro... more Levels of free intracellular calcium have been measured on two cell lines of cultured human fibroblasts carrying the genetic lesions occurring in Duchenne and Becker dystrophies. Both cell lines elicited a markedly higher content of the cation (98 nM and 57 nM, respectively) than control fibroblasts (35 nM). Differences toward controls were statistically significant (p < 0.01). Dystrophic fibroblasts were also found to possess a significantly reduced amount by about 50% of muscular acylphosphatase isoenzyme as compared to normal cells. As acylphosphatase was demonstrated to be involved in the regulation of Ca2+-ATPase activity from different sources, a hypothesis was formulated that could explain the disruption of calcium homeostasis as an effect of the altered acylphosphatase activity.
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