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1.
J Org Chem ; 88(13): 9306-9312, 2023 07 07.
Article in English | MEDLINE | ID: mdl-37314002

ABSTRACT

Herein, we report the total synthesis of nagelamide W (1), a pyrrole imidazole alkaloid of the nagelamide family isolated in 2013. The key approach in this work involves the construction of the 2-aminoimidazoline core of nagelamide W from alkene 6 through a cyanamide bromide intermediate. The synthesis of nagelamide W was accomplished with an overall yield of 6.0%.


Subject(s)
Alkaloids , Pyrroles , Molecular Structure
2.
J Org Chem ; 87(24): 16820-16828, 2022 12 16.
Article in English | MEDLINE | ID: mdl-36475626

ABSTRACT

Here, we report the synthesis of 3,4-disubstituted 1H-pyrazoles and 3,5-disubstituted pyridines from the reaction of epoxides with hydrazine and ammonia, respectively. Both reactions utilize Sc(OTf)3 as a Lewis acid. The pyrazole synthesis utilizes N-bromosuccinimide to convert the intermediate pyrazolines to the pyrazoles, whereas the pyridine synthesis utilizes FeCl3 as a cocatalyst.


Subject(s)
Ammonia , Pyridines , Epoxy Compounds , Pyrazoles , Hydrazines
3.
Biomolecules ; 11(12)2021 11 30.
Article in English | MEDLINE | ID: mdl-34944433

ABSTRACT

The proteasome system is a large and complex molecular machinery responsible for the degradation of misfolded, damaged, and redundant cellular proteins. When proteasome function is impaired, unwanted proteins accumulate, which can lead to several diseases including age-related and neurodegenerative diseases. Enhancing proteasome-mediated substrate degradation with small molecules may therefore be a valuable strategy for the treatment of various neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington's diseases. In this review, we discuss the structure of proteasome and how proteasome's proteolytic activity is associated with aging and various neurodegenerative diseases. We also summarize various classes of compounds that are capable of enhancing, directly or indirectly, proteasome-mediated protein degradation.


Subject(s)
Aging/metabolism , Neurodegenerative Diseases/metabolism , Proteasome Endopeptidase Complex/metabolism , Small Molecule Libraries/pharmacology , Humans , Proteasome Inhibitors/pharmacology , Protein Folding , Proteolysis/drug effects
4.
Acta Physiol (Oxf) ; 210(3): 590-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24119187

ABSTRACT

AIM: In inflamed and damaged cardiovascular tissues, local extracellular adenosine concentrations increase coincidentally with activation of the transcription factor nuclear factor kappa B (NFκB). To investigate whether adenosine influences NFκB activation in vascular smooth muscle cells (VSMCs) and, if so, to examine the role of its receptors. METHODS: VSMCs were isolated from NFκB-luciferase reporter mice, cultured and then treated by lipopolysaccharide (LPS) to activate NFκB signalling. Adenosine, adenosine receptor agonists and antagonists, adenosine deaminase and uptake inhibitors were used together with LPS to evaluate the role of adenosine and its receptors on NFκB activation, which was assessed by luciferase activity and NFκB target gene expression. RESULTS: Adenosine potentiated LPS-induced NFκB activation. This was dependent on adenosine uptake and enhanced by an adenosine deaminase inhibitor, suggesting that intracellular adenosine plays an important role. Non-selective adenosine receptor agonists (2Cl-Ado and NECA) inhibited NFκB activation induced by LPS. Selective A1 or A2A antagonist given alone could not completely antagonize the NECA effect, indicating that the inhibitory effect was due to multiple adenosine receptors. The activation of the A3 receptor further increased LPS-induced NFκB activation. CONCLUSIONS: Adenosine increases LPS-induced nuclear factor kappa B activation in smooth muscle cells via an intracellular mechanism and decreases it via actions on A1 and A2A receptors. These results provide novel insights into the role of adenosine as a regulator of inflammation-induced NFκB activation.


Subject(s)
Adenosine/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , NF-kappa B/metabolism , Receptors, Purinergic P1/metabolism , Animals , Cells, Cultured , Enzyme Activation/physiology , Immunohistochemistry , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
5.
Toxicol In Vitro ; 24(2): 567-77, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19883748

ABSTRACT

In vitro models of tissues, such as the cornea, represent systems for modeling cell-to-cell interactions and tissue function. The objective of this study was to develop an optimized nerve differentiation medium to incorporate into a 3D in vitro model to study innervation and cell targeting. A hybrid neuroblastoma cell line (NDC) was examined for its ability to differentiate into neurons, produce neurites, and functionally contact target cells. Neuronal differentiation of NDCs was optimized through a combinatorial approach which involved culturing cells in the presence of various extracellular matrices and soluble factors. A serum-free medium containing nerve growth factor (NGF), dimethyl sulfoxide (DMSO), or dexamethasone resulted in the greatest proportion of NDCs demonstrating a neuronal morphology. Similarly, with supplementation of cyclic AMP (cAMP) or NGF, neurite extension was optimized. Combining these factors generated an optimized differentiation and extension medium, relative to the individual components alone. In co-culture with epithelial cells, NDC neurites generated in the optimized medium formed contacts with epithelial targets and produced substance P. Similarly, NDCs seeded into a collagen matrix produced neurites that projected through the matrix to target epithelial cells, promoted epithelial stratification, and increased the rate of epithelial wound healing. As well, differentiated NDCs could target and alter acetylcholine receptor clustering in mouse C2C12 myotubes, demonstrating synaptic plasticity. Our data supports the use of NDCs, in combination with optimized medium, for generating an innervated in vitro model.


Subject(s)
Cell Adhesion/drug effects , Cell Differentiation/physiology , Hybrid Cells , Neuroblastoma , Neurons/cytology , Animals , Cell Culture Techniques , Cell Line, Tumor , Chick Embryo , Cyclic AMP/pharmacology , Dexamethasone/pharmacology , Dimethyl Sulfoxide/pharmacology , Ganglia, Spinal/cytology , Mice , Nerve Growth Factor/pharmacology , Neurons/drug effects , Neurons/physiology , Rats , Tissue Culture Techniques
6.
Int J Artif Organs ; 30(7): 619-27, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17674339

ABSTRACT

Hyaline cartilage has very limited regenerative capacity following damage. Therefore engineered tissue substitutes have been the focus of much research. Our objective was to develop a fibrin-based scaffold as a cell delivery vehicle and template for hyaline cartilage regeneration, and compare its cellular properties against monolayer and pellet culture for chondrogenic cells. The chondrogenic precursor cell line, RCJ 3.1C5.18 (C5.18), was chosen as a test system for evaluating the effect of various culture conditions, including cell encapsulation, on articular chondrogenic cell differentiation. The C5.18 cells in monolayer showed elevated expression of collagen II, an articular chondrogenic marker, but also markers for fibrocartilage differentiation (collagen I and versican) when cultured with chondrogenic medium as compared to basic maintenance medium. Pellets of C5.18 cells cultured in chondrogenic medium were histologically more organized in structure than pellets cultured in control maintenance medium. The chondrogenic medium cultured pellets also secreted an extracellular matrix that was comprised of type II with very little type I collagen, indicating a trend towards a more hyaline-like cartilage. Moreover, when cultured in chondrogenic medium, fibrin-encapsulated C5.18 cells elaborated an extracellular matrix containing type II collagen, as well as aggrecan, which are both components of hyaline cartilage. This indicated a more articular-like chondrogenic differentiation for fibrin encapsulated C5.18 cells. The results of these experiments provide evidence that the C5.18 cell line can be used as a tool to evaluate potential scaffolds for articular cartilage tissue engineering.


Subject(s)
Chondrogenesis , Fibrin Tissue Adhesive , Hyaline Cartilage/physiology , Tissue Adhesives , Tissue Engineering/methods , Aggrecans/genetics , Aggrecans/metabolism , Animals , Cell Culture Techniques , Cell Differentiation , Cell Line , Chondrocytes/cytology , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type II/genetics , Collagen Type II/metabolism , Gene Expression Profiling , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Regeneration , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor , Transcription Factors/genetics , Transcription Factors/metabolism , Versicans/genetics , Versicans/metabolism
7.
Acta Physiol (Oxf) ; 190(1): 63-75, 2007 May.
Article in English | MEDLINE | ID: mdl-17428234

ABSTRACT

AIM: To examine cardiac function, body temperature and locomotor behaviour in the awake adenosine A(1) receptor knock out mouse of both sexes. METHODS: Male and female A(1)R (+/+) and (-/-) mice, instrumented with telemetric devices, were recorded during basal conditions and after drug administration. RESULTS: Female mice had higher heart rate, body temperature and locomotion, both during daytime and during the night. Awake A(1)R (-/-) mice had a slightly elevated heart rate, and this was more clear-cut in males. Heart rate was also higher in Langendorff-perfused denervated A(1)R (-/-) hearts. Body temperature was higher in A(1)R (-/-) males and females; locomotor activity was higher in A(1)R (-/-) females, but not in males. The adenosine receptor agonist R-PIA (0.2 mg kg(-1)) decreased heart rate and body temperature, but less in A(1)R (-/-) animals than in A(1)R (+/+) mice (P < 0.001 in both parameters). The unselective adenosine receptor antagonist caffeine had a minor stimulatory effect on heart rate in lower doses, but depressed it at a dose of 75 mg kg(-1). Body temperature was increased after a low dose (7.5 mg kg(-1)) of caffeine in both sexes and genotypes, and markedly reduced after a high dose (75 mg kg(-1)) of caffeine. An intermediary dose of caffeine 30 mg kg(-1) increased or decreased body temperature depending on genotype and sex. Locomotor responses to caffeine were variable depending both on genotype and sex. CONCLUSION: Thus, the adenosine A(1) receptor is involved in the regulation of heart rate, body temperature and locomotor activity, but the magnitude of the involvement is different in males and females.


Subject(s)
Body Temperature/physiology , Heart Rate/physiology , Receptor, Adenosine A1/physiology , Sex Characteristics , Adenosine/analogs & derivatives , Adenosine/pharmacology , Animals , Body Temperature/drug effects , Caffeine/pharmacology , Central Nervous System Stimulants/pharmacology , Female , Genotype , Heart Rate/drug effects , Locomotion/drug effects , Locomotion/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuroprotective Agents/pharmacology , Receptor, Adenosine A1/drug effects , Receptor, Adenosine A1/genetics
8.
Brain Res ; 1098(1): 1-8, 2006 Jul 07.
Article in English | MEDLINE | ID: mdl-16777078

ABSTRACT

The neurotoxic effects of carbon monoxide (CO) are well known. Brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct effect of CO on intracellular targets remains poorly understood. In the present study, we have investigated the pathways leading to neural cell death induced by in vitro exposure to CO using a gas exposure chamber that we have developed. Mouse hippocampal neurons (HT22) and human glial cells (D384) were exposed to concentrations of CO ranging from 300 to 1000 ppm in the presence of 20% oxygen. Cytotoxicity was observed after 48 h exposure to 1000 ppm, corresponding to approximately 1 microM CO in the cultured medium, as measured by gas chromatography. CO induced cell death with characteristic features of apoptosis. Exposed cells exhibited loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, nuclei with chromatin condensation, and exposure of phosphatidyl serine on the external leaflet of the plasma membrane. CO also triggered activation of caspase and calpain proteases. Pre-incubation with either the pancaspase inhibitor Z-VAD-fmk (20 microM) or the calpain inhibitor E64d (25 microM) reduced by 50% the occurrence of apoptosis. When pre-incubating the cells with the two inhibitors together there was an additional reduction in the number of cells with apoptotic nuclei. These data suggest that CO causes apoptosis via activation of parallel proteolytic pathways involving both caspases and calpains. Furthermore, pre-treatment with the antioxidant MnTBAP (100 microM) significantly reduced the number of apoptotic nuclei, pointing to a critical role of oxidative stress in CO toxicity.


Subject(s)
Apoptosis/drug effects , Carbon Monoxide Poisoning/pathology , Hypoxia, Brain/pathology , Neurons/pathology , Animals , Annexin A5/metabolism , Calpain/antagonists & inhibitors , Calpain/metabolism , Carrier Proteins/metabolism , Caspase Inhibitors , Caspases/metabolism , Cell Line , Cell Line, Tumor , Cell Membrane/drug effects , Cell Nucleus/ultrastructure , Culture Media , Cytochromes c/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Hippocampus/pathology , Humans , Immunoblotting , Immunohistochemistry , Membrane Potentials/drug effects , Mice , Microfilament Proteins/metabolism , Mitochondria/drug effects , Neurons/drug effects , Phosphatidylserines/pharmacology , Propidium , Signal Transduction/drug effects , Trypan Blue
9.
Toxicol In Vitro ; 19(1): 11-20, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15582351

ABSTRACT

Styrene-7,8-oxide (SO) is the main metabolite of styrene, a neurotoxic volatile organic compound used industrially. Here we report the novel observation that several markers of oxidative stress were affected in SK-N-MC cells exposed for 16 h to concentrations of SO that induce apoptotic cell death. The production of Thiobarbituric Acid Reactive Substances (TBARS), rose from 69.1 +/- 15.7 nmol/g protein (control) to 119.3 +/- 39.2 and 102.0 +/- 17.3 nmol/g protein after exposure to 0.3 and 1 mM SO, respectively. Carbonyl group levels were significantly enhanced by SO at both concentrations. The lower dose also decreased sulphydryl groups. SO caused a marked oxidative DNA damage, as shown by a fivefold increase in 8-hydroxy-2(')-deoxyguanosine (8-OHdG). In addition, SO exposure resulted in alterations of scavenging abilities, given the reduction of both glutathione (GSH) and glutathione-S-transferase (GST) activity. Induction of expression of the oxidative stress response gene heme-oxygenase-1 (HO-1) and an increased HO-1 activity were also observed. These data provide compelling evidence that oxidative stress significantly contributes to SO toxicity in neuronal cells.


Subject(s)
Carcinogens/toxicity , Deoxyguanosine/analogs & derivatives , Epoxy Compounds/toxicity , Neurons/drug effects , Oxidative Stress/drug effects , 8-Hydroxy-2'-Deoxyguanosine , Biomarkers/metabolism , Cell Line, Tumor , DNA Damage , Deoxyguanosine/metabolism , Dose-Response Relationship, Drug , Free Radical Scavengers/metabolism , Glutathione/metabolism , Glutathione Transferase/metabolism , Humans , Neuroblastoma , Neurons/metabolism , Neurons/pathology , Oxidation-Reduction/drug effects , Proteins/drug effects , Proteins/metabolism , Thiobarbituric Acid Reactive Substances/metabolism
10.
Toxicology ; 201(1-3): 125-32, 2004 Sep 01.
Article in English | MEDLINE | ID: mdl-15297027

ABSTRACT

Styrene 7,8-oxide (SO) is the main metabolite of styrene, a neurotoxic compound used industrially. Neurons exposed to SO undergo apoptosis with characteristic features including chromatin rearrangements and caspase activation. We report that the execution phase of apoptosis induced by SO (0.3 mM) in SK-N-MC neurons is triggered by translocation of apoptogenic factors (e.g., cytochrome c) into the cytosol. In addition, mitochondria exhibit lower Ca2+ capacity and loss of mitochondrial membrane potential (DeltaPsi). Lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), is increased after 12 h. Pre-treatment with the antioxidant MnTBAP (100 microM) prevents the decrease of Ca2+ capacity, cytochrome c release, activation of caspases, exposure of phosphatidylserine and cell death. Hence, the neurotoxic effects of SO are related to mitochondrial damage and oxidative stress.


Subject(s)
Apoptosis/drug effects , Calcium/metabolism , Carcinogens/toxicity , Cytochromes c/metabolism , Epoxy Compounds/toxicity , Mitochondria/drug effects , Neurons/drug effects , Oxidative Stress/drug effects , Humans , Membrane Potentials/drug effects , Mitochondria/metabolism , Neurons/metabolism , Reactive Oxygen Species/pharmacology , Thiobarbituric Acid Reactive Substances/metabolism , Tumor Cells, Cultured
11.
Eur J Neurosci ; 16(10): 2013-6, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12453066

ABSTRACT

We have previously shown that the neurotoxic compounds colchicine, methylmercury (MeHg) and hydrogen peroxide (H2O2) cause apoptosis in primary cultures of cerebellar granule cells (CGC), characterized by nuclear condensation and high-molecular weight DNA fragmentation. However, only colchicine triggers the activation of caspases, suggesting that factors other than caspase-activated DNase (CAD) are responsible for DNA cleavage in the other two models. Here we report that the two agents that cause oxidative stress, MeHg (1 micro m) and H2O2 (50 micro m), induce translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus in CGC. Our data suggest that, in absence of caspase activity, AIF translocation could be a key event leading to chromatin condensation and DNA degradation in CGC exposed to MeHg and H2O2.


Subject(s)
Cell Nucleus/metabolism , Cerebellum/metabolism , Flavoproteins/metabolism , Granulocytes/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Oxidants/adverse effects , Oxidative Stress , Animals , Apoptosis , Apoptosis Inducing Factor , Cell Culture Techniques , Cerebellum/cytology , Cerebellum/drug effects , DNA Fragmentation , Flavoproteins/drug effects , Granulocytes/drug effects , Granulocytes/ultrastructure , Hydrogen Peroxide/adverse effects , Membrane Proteins/drug effects , Methylmercury Compounds/adverse effects , Rats , Rats, Sprague-Dawley
12.
J Neural Transm (Vienna) ; 109(5-6): 597-605, 2002 May.
Article in English | MEDLINE | ID: mdl-12111452

ABSTRACT

Methylmercury cation (MeHg) is an hazardous environmental pollutant with neurotoxic action. Little is known about the effects of MeHg on catecholaminergic neurons. In the present study we have used epifluorescence microscopy and confocal microscopy to investigate the alterations induced by MeHg in primary DA (dopaminergic) cells isolated from the ventral mesencephalon of CD-1 embryonic mice and cultured for six days in vitro. DA cells were identified in the multi-culture by immunocytochemistry using a tyrosine-hydroxylase antibody. The morphometric analysis of DA neurons exposed to 1 microM MeHg demonstrated a striking decrease in the number of neurites, indicative of cytoskeletal alteration. In addition, DA neurons displayed cell shrinkage and a significant increase of nuclei with chromatin condensation. Based on these results it is concluded that MeHg is highly toxic to primary DA neurons.


Subject(s)
Dopamine/metabolism , Mesencephalon/drug effects , Mesencephalon/metabolism , Methylmercury Compounds/pharmacology , Nerve Degeneration/chemically induced , Neurites/drug effects , Animals , Cell Nucleus/diagnostic imaging , Cell Nucleus/drug effects , Cell Size , Cells, Cultured , DNA Damage , Embryo, Mammalian , Mesencephalon/pathology , Mice , Mice, Inbred Strains , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Neurites/physiology , Neurons/pathology , Ultrasonography
13.
Free Radic Biol Med ; 30(12): 1347-56, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11390179

ABSTRACT

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 microM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H(2)O(2)) exposure (100 microM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H(2)O(2) preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H(2)O(2) stimulated divergent pathways, with caspases being activated only by H(2)O(2). The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H(2)O(2), suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H(2)O(2). The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.


Subject(s)
Apoptosis/drug effects , Astrocytes/drug effects , Hydrogen Peroxide/pharmacology , Lysosomes/drug effects , Methylmercury Compounds/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Astrocytes/ultrastructure , Astrocytoma/pathology , Brain Neoplasms/pathology , Caspases/metabolism , Cell Membrane Permeability/drug effects , Cyclosporine/pharmacology , Cysteine Proteinase Inhibitors/metabolism , DNA Fragmentation/drug effects , Deoxyribonucleases/metabolism , Enzyme Activation/drug effects , Humans , Hydrogen Peroxide/toxicity , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Lysosomes/enzymology , Lysosomes/ultrastructure , Membrane Potentials/drug effects , Methylmercury Compounds/toxicity , Oxidative Stress , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/ultrastructure
14.
Environ Toxicol Pharmacol ; 9(3): 61-70, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11167150

ABSTRACT

We have shown previously that prenatal exposure of rats to 0.5 mg/kg/day of methylmercury (MeHg) produces gender-dependent changes in motor activity in adulthood. In the present study we have investigated whether changes in motor activity could also be found during early ontogeny of the offspring. Pregnant rats were treated with MeHg from day 7 of pregnancy to day 7 of lactation. The habituation to a novel environment (spontaneous activity) and the response to stimulation of the dopaminergic system were studied on postnatal day 14 and 21. Measures of spontaneous activity showed a slight increase in MeHg-prenatal exposed male and female rats at 14 days, but not at 21 days. Following administration of U91356A, a selective dopamine D(2) receptor agonist, a significantly lower dopamine-mediated locomotor activity was observed in the 21 day old MeHg-treated males, but not in females. These results show that prenatal exposure to MeHg alters postjunctional dopaminergic activity during the period of maturation of the dopamine system in the brain. Moreover, the gender-dependent susceptibility previously found in adulthood is already evident at the prepubertal stage.

15.
Neurotox Res ; 3(5): 501-14, 2001 Oct.
Article in English | MEDLINE | ID: mdl-14715461

ABSTRACT

The death of a cell via apoptosis is characterized by morphological changes including cell shrinkage and nuclear condensation. Intracellularly, proteases, including caspases, are activated. In the present article we have compared the ability of three different neurotoxic agents to induce caspase activity in cerebellar granule cells (CGC). These compounds are the microtubule-disrupting agent colchicine and the oxidative stress-inducing agents hydrogen peroxide and methylmercury (MeHg). We have previously shown that each of these agents causes nuclear changes that are consistent with apoptosis, i.e., induction of chromatin condensation and DNA cleavage into fragments of regular size (700, 300 and 50 kbp). However, only colchicine causes a large increase in caspase activity, as monitored by the ability of whole cell extracts to cleave the synthetic caspase substrate DEVD-MCA. In contrast, MeHg and hydrogen peroxide do not induce any significant increase of DEVDase activity as compared to control cells. Immunocytochemistry confirms that active caspase-3 is abundant only in colchicine-exposed cells. In agreement with these findings, the pan-caspase inhibitor, z-VAD-fmk, is efficient in protecting CGC against colchicine, but not against hydrogen peroxide or MeHg. These data suggest that in CGC the activation of caspases is not always required to induce morphological changes and pattern of DNA fragmentation consistent with apoptosis.

16.
J Neurosci Res ; 62(4): 557-65, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11070499

ABSTRACT

Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well-known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 microM to 1.5 microM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high-molecular-weight DNA fragments. Nevertheless, caspase-3-like activity was not significantly induced, and the broad caspase inhibitor Z-VAD-FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg-induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase-3 and alpha-fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17beta-estradiol (10 microM) and the Delta(8,9)-dehydro derivative of 17alpha-estradiol J811 (10 microM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17beta-estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication.


Subject(s)
Antioxidants/pharmacology , Apoptosis/drug effects , Estradiol/pharmacology , Free Radical Scavengers/pharmacology , Methylmercury Compounds/toxicity , Neurons/drug effects , Neuroprotective Agents/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Animals, Newborn , Apoptosis/physiology , Calpain/drug effects , Calpain/metabolism , Caspase 3 , Caspases/drug effects , Caspases/metabolism , Cell Size/drug effects , Cell Size/physiology , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cerebellum/cytology , Cerebellum/drug effects , Cerebellum/metabolism , Drug Interactions/physiology , Environmental Exposure/adverse effects , Female , Neurons/cytology , Neurons/metabolism , Neurotoxins/toxicity , Pregnancy , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology
17.
J Mol Endocrinol ; 21(1): 7-17, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9723859

ABSTRACT

The regulation of parathyroid hormone secretion by the chief cells of the parathyroid is mediated by a 7-transmembrane (7-TM) Ca2+-sensing receptor (CaR), which signals via activation of pertussis toxin-insensitive G proteins, causing stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC). We have identified the PI-PLC isoforms expressed in two model systems utilized for studying CaR signal transduction, i.e. dispersed bovine parathyroid cells and a human embryonic kidney cell line (HEK 293) stably transfected with the human parathyroid CaR-cDNA. All of the eight PI-PLC isozymes examined in this study were found to be expressed to varying extents in the bovine parathyroid gland and in the CaR-transfected HEK cells as assessed by immunoblotting. We localized the expression of the more abundant isozymes (beta1, beta2, beta3, gamma1, gamma2, delta2) to the chief cells of the bovine parathyroid by immunocytochemistry, while the two less abundant isozymes (delta1, beta4) were not detectable in parathyroid sections. G proteins activated by 7-TM receptors are known to activate mainly PI-PLC of the beta class. Therefore, beta1, beta2, beta3 and beta4, all expressed in the bovine parathyroid, are candidate isozymes for coupling to the CaR. A comparison of the levels of expression of PI-PLC isozymes between CaR-transfected HEK cells and non-transfected HEK cells suggested that the expression of the CaR in this human cell line does not cause a significant up-regulation of any of the PLCbeta and PLCgamma isozymes. PLCdelta2, showing predominantly nuclear localization in the parathyroid, was the sole PI-PLC isozyme with higher levels of expression in CaR-transfected HEK cells.


Subject(s)
Isoenzymes/metabolism , Kidney/enzymology , Parathyroid Glands/enzymology , Receptors, Cell Surface/genetics , Type C Phospholipases/metabolism , Animals , Blotting, Western , Cattle , Cell Line , Humans , Immunohistochemistry , Kidney/metabolism , Parathyroid Glands/metabolism , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Receptors, Calcium-Sensing , Transfection
18.
Somat Cell Mol Genet ; 22(3): 201-10, 1996 May.
Article in English | MEDLINE | ID: mdl-8914605

ABSTRACT

Sequencing of hprt cDNA revealed that three spontaneous mutants in V79 Chinese hamster cells exhibit tandem duplications of exon(s), i.e., either exons 2 and 3 or exon 7. Sequences of different sizes (4.5-8 Kb) were found to be duplicated and inserted in tandem into the hprt gene. These mutants demonstrated spontaneous reversion frequencies which were about 40-fold higher than those observed with other types of spontaneous mutants, but on the same order of magnitude as spontaneous reversions in Sp5, a mutant with a duplication insertion involving exon 2 in this gene. These data suggest that all of the duplications found have the same genetic instability, regardless of the type, size or position of the duplicated fragment. The coding sequence of the hprt cDNA and the restriction pattern of the revertants were virtually identical to the wild-type, indicating restoration of a functional hprt gene by precise deletion of the duplicated fragment.


Subject(s)
Hypoxanthine Phosphoribosyltransferase/genetics , Multigene Family , Animals , Blotting, Southern , Cell Line , Cricetinae , Cricetulus , DNA, Complementary/genetics , Exons/genetics , Mutation , Sequence Analysis, DNA
19.
J Mol Biol ; 252(5): 514-21, 1995 Oct 06.
Article in English | MEDLINE | ID: mdl-7563070

ABSTRACT

dCMP-deaminase-deficient V79/dC hamster cells have highly imbalanced deoxyribonucleoside triphosphate (dNTP) pools, i.e. a 17-fold larger dCTP pool, a slightly reduced dTTP and a very low dGTP pool, compared to dCMP-deaminase-proficient V79/p cells. Nevertheless, the two lines showed the same rates of spontaneous mutation at the hprt and ouabain-resistance loci. Analysis of spontaneous hprt mutations indicated an increase in misincorporation of C in V79/dC cells, although it was not statistically significant. When the dCTP pool was further increased fivefold by incubating V79/dC cells with cytidine, C misincorporation increased to 88%, but the mutation frequency remained unchanged. The dNTP pools of V79/dC cells were also altered by treatment with thymidine, or with thymidine plus deoxycytidine. After incubation with thymidine alone, the dCTP pool all but disappeared, whereas it maintained a normal level in the presence of deoxycytidine. In both cases dTTP rose to nmol amounts, and dGTP accumulated. Incubation with 10 mM thymidine was the only treatment that increased the mutation frequency; T misincorporation then accounted for 94% of the base substitutions. In the presence of deoxycytidine the cells had a dTTP/dCTP ratio of 0.04, but 86% of the base substitutions involved C misincorporation and most probably originated from G mis-incorporation caused by excess dGTP. Alterations of RNA splicing and hot spots for base substitutions varied with the imbalance, the latter showed "next-nucleotide effects". Our results suggest that the fidelity of DNA replication in V79 cells is only affected by large changes in the pool and is more sensitive to changes in dGTP than in dCTP or dTTP.


Subject(s)
Deoxycytosine Nucleotides/metabolism , Deoxyguanine Nucleotides/metabolism , Hypoxanthine Phosphoribosyltransferase/genetics , Mutation , Thymine Nucleotides/metabolism , Animals , Cell Line , Cricetinae , Cytidine/metabolism , DCMP Deaminase/deficiency , DCMP Deaminase/genetics , DCMP Deaminase/metabolism , DNA Replication/genetics , Fibroblasts , Thymidine/metabolism
20.
Genomics ; 26(3): 467-72, 1995 Apr 10.
Article in English | MEDLINE | ID: mdl-7607669

ABSTRACT

We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C beta 3 (PLC beta 3) gene (gene symbol PLCB3). PLC beta 3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC beta 3, previously published, was extended with 876 bp in the 5' direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5'flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters.


Subject(s)
Isoenzymes/genetics , Type C Phospholipases/genetics , Amino Acid Sequence , Base Sequence , DNA, Complementary , Exons , Humans , Introns , Molecular Sequence Data , Phospholipase C beta , RNA Splicing , Transcription, Genetic
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