Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 29
Filter
Add more filters










Publication year range
1.
J Dent Res ; 96(5): 509-515, 2017 May.
Article in English | MEDLINE | ID: mdl-28107637

ABSTRACT

This work establishes a survival probability methodology for interface-initiated fatigue failures of monolithic ceramic crowns under simulated masticatory loading. A complete 3-dimensional (3D) finite element analysis model of a minimally reduced molar crown was developed using commercially available hardware and software. Estimates of material surface flaw distributions and fatigue parameters for 3 reinforced glass-ceramics (fluormica [FM], leucite [LR], and lithium disilicate [LD]) and a dense sintered yttrium-stabilized zirconia (YZ) were obtained from the literature and incorporated into the model. Utilizing the proposed fracture mechanics-based model, crown survival probability as a function of loading cycles was obtained from simulations performed on the 4 ceramic materials utilizing identical crown geometries and loading conditions. The weaker ceramic materials (FM and LR) resulted in lower survival rates than the more recently developed higher-strength ceramic materials (LD and YZ). The simulated 10-y survival rate of crowns fabricated from YZ was only slightly better than those fabricated from LD. In addition, 2 of the model crown systems (FM and LD) were expanded to determine regional-dependent failure probabilities. This analysis predicted that the LD-based crowns were more likely to fail from fractures initiating from margin areas, whereas the FM-based crowns showed a slightly higher probability of failure from fractures initiating from the occlusal table below the contact areas. These 2 predicted fracture initiation locations have some agreement with reported fractographic analyses of failed crowns. In this model, we considered the maximum tensile stress tangential to the interfacial surface, as opposed to the more universally reported maximum principal stress, because it more directly impacts crack propagation. While the accuracy of these predictions needs to be experimentally verified, the model can provide a fundamental understanding of the importance that pre-existing flaws at the intaglio surface have on fatigue failures.


Subject(s)
Ceramics/chemistry , Crowns , Dental Restoration Failure , Computer Simulation , Dental Porcelain/chemistry , Dental Stress Analysis , Finite Element Analysis , Probability
2.
Physiol Res ; 52(4): 481-96, 2003.
Article in English | MEDLINE | ID: mdl-12899662

ABSTRACT

The development of the cauda equina syndrome in the dog and the involvement of spinal nitric oxide synthase immunoreactivity (NOS-IR) and catalytic nitric oxide synthase (cNOS) activity were studied in a pain model caused by multiple cauda equina constrictions. Increased NOS-IR was found two days post-constriction in neurons of the deep dorsal horn and in large, mostly bipolar neurons located in the internal basal nucleus of Cajal seen along the medial border of the dorsal horn. Concomitantly, NOS-IR was detected in small neurons close to the medioventral border of the ventral horn. High NOS-IR appeared in a dense sacral vascular body close to the Lissauer tract in S1-S3 segments. Somatic and fiber-like NOS-IR appeared at five days post-constriction in the Lissauer tract and in the lateral and medial collateral pathways arising from the Lissauer tract. Both pathways were accompanied by a dense punctate NOS immunopositive staining. Simultaneously, the internal basal nucleus of Cajal and neuropil of this nucleus exhibited high NOS-IR. A significant decrease in the number of small NOS immunoreactive somata was noted in laminae I-II of L6-S2 segments at five days post-constriction while, at the same time, the number of NOS immunoreactive neurons located in laminae VIII and IX was significantly increased. Moreover, high immunopositivity in the sacral vascular body persisted along with a highly expressed NOS-IR staining of vessels supplying the dorsal sacral gray commissure and dorsal horn in S1-S3 segments. cNOS activity, based on a radioassay of compartmentalized gray and white matter regions of lower lumbar segments and non-compartmentalized gray and white matter of S1-S3 segments, proved to be highly variable for both post-constriction periods.


Subject(s)
Nitric Oxide Synthase/metabolism , Polyradiculopathy/enzymology , Spinal Cord/enzymology , Animals , Arginine/metabolism , Catalysis , Citrulline/metabolism , Dogs , Female , Immunohistochemistry , Male , NADPH Dehydrogenase/metabolism , Nitric Oxide Synthase Type I , Radioimmunoassay
3.
J Mater Sci Mater Med ; 14(1): 17-26, 2003 Jan.
Article in English | MEDLINE | ID: mdl-15348534

ABSTRACT

This paper presents a combined experimental and computational study of the influence of indenter ball size on contact damage in model multilayered structures with equivalent elastic properties to bonded dentin/crown structures. Following a brief description of restored tooth structures, prior work on the development of model dental multilayered structures is reviewed. The effects of indentation ball size are investigated within a combined experimental and computational/analytical framework. The observed cracking patterns at the onset of crack nucleation are shown to be associated with principal stress contours computed using finite element analysis. The implications of the results are discussed for the design of dental multilayers that are more resistant to crack nucleation and propagation.

4.
Bioorg Med Chem Lett ; 11(16): 2067-70, 2001 Aug 20.
Article in English | MEDLINE | ID: mdl-11514140

ABSTRACT

The synthesis and structure-activity relationship (SAR) study of a novel series of N-type calcium channel blockers are described. L-Cysteine derivative 2a was found to be a potent and selective N-type calcium channel blocker with IC(50) 0.63 microM on IMR-32 assay. Compound 2a showed analgesic efficacy in the rat formalin-induced pain model by intrathecal and oral administration.


Subject(s)
Amino Acids/pharmacology , Analgesics/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, N-Type/metabolism , Amino Acids/chemical synthesis , Amino Acids/chemistry , Amino Acids/therapeutic use , Analgesics/chemical synthesis , Analgesics/chemistry , Analgesics/therapeutic use , Animals , Calcium Channel Blockers/chemical synthesis , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/therapeutic use , Calcium Channels, N-Type/drug effects , Disease Models, Animal , Formaldehyde , Pain/chemically induced , Pain/drug therapy , Rats , Structure-Activity Relationship
5.
Prog Neurobiol ; 64(6): 613-37, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11311464

ABSTRACT

Single or double-level compression of the lumbosacral nerve roots located in the dural sac results in a polyradicular symptomatology clinically diagnosed as cauda equina syndrome. The cauda equina nerve roots provide the sensory and motor innervation of most of the lower extremities, the pelvic floor and the sphincters. Therefore, in a fully developed cauda equina syndrome, multiple signs of sensory disorders may appear. These disorders include low-back pain, saddle anesthesia, bilateral sciatica, then motor weakness of the lower extremities or chronic paraplegia and, bladder dysfunction. Multiple etiologies can cause the cauda equina syndrome. Among them, non-neoplastic compressive etiologies such as herniated lumbosacral discs and spinal stenosis and spinal neoplasms play a significant role in the development of the cauda equina syndrome. Non-compressive etiologies of the cauda equina syndrome include ischemic insults, inflammatory conditions, spinal arachnoiditis and other infectious etiologies. The use of canine, porcine and rat models mimicking the cauda equina syndrome enabled discovery of the effects of the compression on nerve root neural and vascular anatomy, the impairment of impulse propagation and the changes of the neurotransmitters in the spinal cord after compression of cauda equina. The involvement of intrinsic spinal cord neurons in the compression-induced cauda equina syndrome includes anterograde, retrograde and transneuronal degeneration in the lumbosacral segments. Prominent changes of NADPH diaphorase exhibiting, Fos-like immunoreactive and heat shock protein HSP72 were detected in the lumbosacral segments in a short-and long-lasting compression of the cauda equina in the dog. Developments in the diagnosis and treatment of patients with back pain, sciatica and with a herniated lumbar disc are mentioned, including many treatment options available.


Subject(s)
Cauda Equina/physiology , Disease Models, Animal , Nerve Compression Syndromes/physiopathology , Polyradiculopathy/physiopathology , Animals , Cauda Equina/blood supply , Humans , Nerve Compression Syndromes/diagnosis , Nerve Compression Syndromes/therapy , Polyradiculopathy/diagnosis , Polyradiculopathy/therapy
6.
J Neurosci Methods ; 104(2): 191-8, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11164245

ABSTRACT

In the present study we characterize a rat neurogenic intermittent claudication model which was accomplished by placing two pieces of silicone rubber of various sizes into the lumbar (L4 and L6) epidural space. After induction of spinal stenosis walking function was measured using a treadmill apparatus and sensory functions were tested by measuring thermal and tactile withdrawal threshold (von Frey filaments) for the period of 28 days after stenosis. In addition, local spinal cord blood flow (SCBF) was measured, periodically, before and after induction of stenosis using laser Doppler. After implantation of two pieces of silicone rubber (width 1.25 mm, height 1.0 mm, length 4.0 mm) a significant running dysfunction, as evidenced by shortening of running distance, was measured as soon as 24 h after stenosis (178.5+/-59.1 m vs 681.3+/-70.2 m). This effect persisted for 28 days after surgery. Similarly, a significant tactile (but not thermal) hypersensitivity was measured for a period of 28 days (1.2+/-0.3 g vs 14.9+/-0.2 g). In this experimental group the measurement of local SCBF revealed a significant (30-50%) reduction in the territory of spinal stenosis measured at 3,7,14 or 28 days after surgery. Implantation of larger pieces of silicon rubber (1.5 mm width) caused a significant increase in the incidence of urinary retention and mortality rate. These data show that chronic partial spinal compression at L4 and L6 spinal level lead to the development of significant motor/sensory dysfunction which resemble those seen in patients with neurogenic intermittent claudication. The lack of motor dysfunction under resting conditions but its appearance during forced exercise also suggest that the development of local spinal ischemia can represent one of the mechanisms.


Subject(s)
Disease Models, Animal , Intermittent Claudication/physiopathology , Animals , Cauda Equina/physiopathology , Exercise Test , Exploratory Behavior/physiology , Locomotion/physiology , Male , Motor Activity/physiology , Nerve Compression Syndromes/physiopathology , Rats , Rats, Wistar , Running/physiology , Sensory Thresholds/physiology , Spinal Cord/blood supply , Spinal Stenosis/physiopathology , Touch/physiology
7.
J Invest Dermatol ; 117(6): 1621-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11886531

ABSTRACT

Intradermal injection of substance P elicits an itch sensation in human subjects and an itch-associated response in mice. The substance P-induced itch-associated response in mice is not inhibited by antihistamine. Therefore, the mechanisms of substance P-induced itch-associated response are unclear. In this study, we demonstrated one of the mechanisms. Substance P induces an arachidonate cascade to produce prostaglandins and leukotriene. In this study we considered whether arachidonate metabolites are involved in the substance P-induced itch-associated response. A phospholipase A(2) inhibitor arachidoryltrifluoromethyl ketone inhibited the substance P-induced itch-associated response in mice. Pre treatment with the glucocorticoids betamethasone and dexamethasone also produced inhibition of the substance P-induced itch-associated response in mice as well as humans. The 5-lipoxygenase inhibitor zileuton, but not the cyclooxygenase inhibitors indomethacin and diclofenac, suppressed substance P-induced itch-associated response. The leukotriene B(4) receptor antagonist 5-[2-(2-carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]valeric acid produced inhibition, whereas pranlukast (leukotriene C(4)/D(4)/E(4) receptor antagonist) and 5(Z)-7-[1S,2S, 3S,5R-3-(trans-b-styren)sulfonamido-6,6-dimethylbi cyclo(3,1,1)hept-2-yl]-5-heptenoic acid (EP(1) receptor antagonist) were without effect. Furthermore, when the production of leukotriene B(4) and prostaglandin E(2) was measured in skin injected with substance P and in mouse keratinocytes applied with substance P, the level of both products increased. As leukotriene B(4), but not prostaglandin E(2), also induces the itch-associated response in mice, these results suggest that leukotriene B(4) and keratinocytes, cutaneous cells which produced leukotriene B(4), play an important role in substance P-induced itch-scratch response in mice. Leukotriene B(4) receptor antagonist and 5-lipoxygenase inhibitor may be novel antipruritic drugs.


Subject(s)
Keratinocytes/immunology , Leukotriene B4/immunology , Pruritus/immunology , Substance P/pharmacology , Animals , Arachidonate 5-Lipoxygenase/metabolism , Arachidonic Acid/metabolism , Arachidonic Acids/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Dexamethasone/pharmacology , Dinoprostone/metabolism , Enzyme Inhibitors/pharmacology , Glucocorticoids/pharmacology , Immunosuppressive Agents/pharmacology , Indomethacin/pharmacology , Keratinocytes/metabolism , Leukotriene B4/metabolism , Lipoxygenase Inhibitors , Male , Mast Cells , Mice , Mice, Inbred ICR , Mice, Mutant Strains , Phenylpropionates/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Pruritus/chemically induced , Receptors, Leukotriene B4/antagonists & inhibitors , Receptors, Leukotriene B4/metabolism , Receptors, Neurokinin-1/metabolism
8.
J Inorg Biochem ; 82(1-4): 239-49, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11132634

ABSTRACT

A zinc (II) complex with a macrocyclic tetraamine appended with an anthraquinone ((9,10-anthraquinon-2-yl)methyl-1,4,7,10-tetraazacyclododecane, ZnL, anthraquinonyl-cyclen) selectively recognizes consecutive G sequence in double-stranded DNA. The affinity of the Zn2+-anthraquinonyl-cyclen to consecutive dG groups in DNA was disclosed by comparison of K(app) values (=[DNA-bound ZnL]/[uncomplexed ZnL][uncomplexed nucleobase in DNA]) determined by the UV spectrophotometric titrations at pH 8, I=0.1 (NaNO3), and 25 degrees C for poly(dG) x poly(dC) (K(app) = 1.5 x 10(5) M(-1)), poly(dG-dC)2 (2.8 x 10(4) M(-1)), poly(dA-dT)2 (4.3 x 10(4) M(-1)), and calf thymus DNA (2.8 x 10(4) M(-1)). The corresponding K(app) values with the Zn2+-free ligand were 5.3 x 10(3) M(-1), 7.4 x 10(3) M(-1), 7.4 x 10(3) M(-1), and 5.9 x 10(3) M(-1), respectively. The selective recognition of consecutive G sequence was concluded from the DNase I footprinting of SV40 early promotor DNA fraction (197 bp) containing a TATA box and six GC boxes. The present finding is in remarkable contrast to the previous selective T-recognition by Zn2+-cyclen complexes appended with acridine, quinoline(s), and naphthalene(s) [J. Am. Chem. Soc. 121 (1999) 5426]. While the Zn2+-acridinyl-cyclen inhibited TATA binding protein from interacting with a TATA box consensus DNA [J. Inorg. Biochem. 79 (2000) 253], the present Zn2+-anthraquinonyl-cyclen inhibited the Sp1 transcriptional factor protein from interacting with a GC box-consensus DNA.


Subject(s)
Anthraquinones/chemistry , DNA/chemistry , Guanine/analysis , Organometallic Compounds/chemistry , Zinc/chemistry , Anthraquinones/metabolism , Base Sequence , DNA/metabolism , DNA Footprinting , Ethidium/chemistry , Guanine/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , Molecular Structure , Organometallic Compounds/metabolism , Regulatory Sequences, Nucleic Acid , Spectrophotometry
9.
J Biol Inorg Chem ; 4(4): 431-40, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10555577

ABSTRACT

The characteristic binding mode of zinc(II) complexes of macrocyclic tetraamines (1,4,7,10-tetraazacyclododecane, cyclen) appended with one or two arylmethyl group(s) [(4-quinolyl)methyl-, 1,7-bis(4-quinolyl)methyl-, (1-naphthyl)methyl-, 1,7-bis(1-naphthyl)methyl-, and (9-acridinyl)methyl-cyclen] to double-stranded calf thymus DNA and synthetic DNAs [poly(dA)-poly(dT), poly(dA-dT)2, poly(dI).poly(dC), poly(dI-dC)2, poly(dG) poly(dC), and poly(dG-dC)2] has been examined by spectrophotometric methods, Tm measurement, and inhibition of these DNA-directed transcriptions in vitro. Various hypochromic and bathochromic effects on the pendant aromatic absorption spectra of the complexes were observed in titration with the native and synthetic DNA. The binding constants Kapp (=[bound cyclen derivatives]/[unbound cyclen derivatives][DNA phosphates] M(-1)), at 25 degrees C in 10 mM EPPS (pH 8.0) containing 0.1 M Na+, were determined and compared with those of the corresponding Zn2+ -free ligands. The results showed that the Zn2+ -cyclen complexes interact with the DNA more strongly than the corresponding diprotonated ligands, leading to a stronger stacking of the pendant aromatic rings. The binding of Zn2+ -(9-acridinyl)methyl-cyclen to calf thymus DNA was competed by an AT-selective, minor groove binder, distamycin, but not by a major groove binder, methyl green. In an unusual interaction of excess Zn2+ -(9-acridinyl)methyl-cyclen with poly(dA).poly(dT), the Zn2+ -cyclen moiety went into the minor groove to make coordination bonds with the deprotonated imides of the thymines, resulting in disruption of the poly(dA).poly(dT) duplex. Thymine-containing DNA-directed transcription with Escherichia coli RNA polymerase in vitro was inhibited by the Zn2+ -(9-acridinyl)methyl-cyclen. The 50% inhibition concentrations of the transcription (IC50) were 22-45 microM with poly(dA).poly(dT) or poly(dA-dT)2 as templates, while with poly(dG-dC)2 as a template the IC50 value was 110 microM.


Subject(s)
DNA/metabolism , Heterocyclic Compounds, 1-Ring/chemistry , Heterocyclic Compounds, 1-Ring/metabolism , Organometallic Compounds/chemistry , Organometallic Compounds/metabolism , Zinc Compounds/chemistry , Zinc Compounds/metabolism , Amines/chemistry , Amines/metabolism , Animals , Cattle , Cyclams , DNA/chemical synthesis , DNA/drug effects , DNA-Directed RNA Polymerases/metabolism , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/metabolism , Inhibitory Concentration 50 , Poly U/metabolism , Poly dA-dT/chemistry , Poly dA-dT/metabolism , Spectrophotometry, Ultraviolet , Templates, Genetic , Thymus Gland/physiology , Transcription, Genetic , Zinc Compounds/pharmacology
10.
Neuroreport ; 10(10): 2029-33, 1999 Jul 13.
Article in English | MEDLINE | ID: mdl-10424669

ABSTRACT

The cDNAs for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) were cloned from cerebellar neurons undergoing age-induced apoptosis and/or healthy cells. COS-7 cells were transfected with the isolated GAPDH cDNAs using to the Lipofectamine method. Assessment of cell death in this paradigm was performed by monitoring the co-transfected luciferase activities and the characterization of cell death was examined by the DNA fragmentation assay and Hoechst dye nuclear staining. These observations show that over-expression of GAPDH occurring from both cDNAs robustly induces apoptotic death in the transfected COS-7 cell cultures. Confocal-immunocytochemical studies using this GAPDH-specific monoclonal antibody revealed that nuclear translocation of overexpressed GAPDH is a primary apoptotic event. Our results directly demonstrate that over-expressed GAPDH functions as a 'killing protein' in apoptosis.


Subject(s)
Apoptosis/physiology , DNA, Complementary/genetics , Gene Expression Regulation/physiology , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Transfection , Animals , COS Cells , Cloning, Molecular , Rats , Rats, Sprague-Dawley
11.
J Pharmacol Exp Ther ; 288(1): 6-13, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9862746

ABSTRACT

Primary cultures of rat cerebral cortical cells and cerebellar granule cells die by an apoptotic mechanism after more than 2 weeks in cultures in the absence of medium change and glucose supplement, a process termed age-induced apoptosis of cultured neurons. Our preliminary study has shown that age-induced apoptosis of cerebellar granule cells is protected by pretreatment with tetrahydroaminoacridine (THA), an antidementia drug. In this study, we systematically compared the neuroprotective effects of THA with those of (S)-1-[N-(4-chlorobenzyl)succinamoyl]pyrrolidine-2-carbaldehyde (ONO-1603), a novel prolyl endopeptidase inhibitor and potential antidementia drug. Both ONO-1603 and THA effectively delay age-induced apoptosis of cerebral and cerebellar neurons, as demonstrated morphologically with toluidine blue and fluorescein diacetate/propidium iodide staining or biochemically by DNA laddering analysis on agarose gels. ONO-1603 is about 300 times more potent than THA, with a maximal protective effect at 0.03 and 10 microM, respectively. ONO-1603 shows a wide protective range of 0.03 to 1 microM in contrast to a narrow effective range of 3 to 10 microM for THA. Moreover, ONO-1603 is nontoxic to neurons, even at the high concentration of 100 microM, whereas THA elicits severe neurotoxicity at a dose of >/=30 microM. Both ONO-1603 and THA robustly suppress overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) mRNA and accumulation of GAPDH protein in a particulate fraction of cultured neurons undergoing age-induced apoptosis. Because we documented that GAPDH overexpression participates in neuronal apoptosis induced by various insults, we conclude that the neuroprotective actions of ONO-1603 and THA appear to be mediated by suppression of this protein overexpression.


Subject(s)
Apoptosis/drug effects , Gene Expression/drug effects , Glyceraldehyde-3-Phosphate Dehydrogenases/biosynthesis , Neurons/drug effects , Neuroprotective Agents/pharmacology , Pyrrolidines/pharmacology , Animals , Cells, Cultured , Cellular Senescence , Central Nervous System/drug effects , Central Nervous System/enzymology , Central Nervous System/pathology , Cholinesterase Inhibitors/pharmacology , Dementia/drug therapy , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Neurons/enzymology , Neurons/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tacrine/pharmacology
12.
J Pharmacol Exp Ther ; 286(1): 539-47, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9655900

ABSTRACT

We have studied the neuroprotective actions of lithium against various insults in cultured cerebellar granule cells of rats. The anticonvulsants, phenytoin and carbamazepine, have been shown to induce apoptosis of cerebellar granule cells at high concentrations. Here we found that co-presence of LiCl (1-10 mM) dose-dependently protected against phenytoin (20 microM)- and carbamazepine (100 microM)-induced neuronal apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism, morphological inspection, chromatin condensation and DNA fragmentation. These neuroprotective effects were not prevented by inclusion of myoinositol nor mimicked by a potent inositol monophosphatase inhibitor, suggestive of a mechanism independent of inositol monophosphatase blockade. Lithium also significantly protected against apoptosis of cerebellar granule cells induced by aging of the cultures. Additionally, lithium suppressed death of cerebellar granule cells exposed to a low concentration of extracellular potassium. In contrast, it had no protective effect on cell death induced by Ca++ ionophores, a Na+ channel opener, a protein kinase inhibitor, a nitric oxide donor or H2O2. Thus, lithium has robust neuroprotective effects against apoptotic cell death induced by multiple insults with limited selectivity. These actions provide a new avenue to study the molecular and cellular mechanisms of this drug.


Subject(s)
Anticonvulsants/toxicity , Carbamazepine/toxicity , Cerebellum/drug effects , Lithium/pharmacology , Neuroprotective Agents/pharmacology , Phenytoin/toxicity , 5'-Nucleotidase/antagonists & inhibitors , Animals , Cells, Cultured , Inositol/pharmacology , Phosphatidylinositol 3-Kinases/physiology , Potassium/pharmacology , Rats , Rats, Sprague-Dawley
13.
Mol Pharmacol ; 53(4): 701-7, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9547361

ABSTRACT

We recently reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is directly involved in cytosine arabinonucleoside (ara-C)- and low K+-induced neuronal death of cultured cerebellar granule cells. The former is entirely due to apoptosis, whereas the latter involves both apoptosis and necrosis. We examined the subcellular distribution of the overexpressed GAPDH occurring during apoptosis by using both subcellular fractionation and immunocytochemistry with a monoclonal antibody directed against this overexpressed protein. When immature cerebellar neurons were exposed to ara-C, an overexpression of GAPDH was observed, primarily in the nuclear fraction. In contrast, low K+ exposure of mature cerebellar neurons induced the overexpression of GAPDH not only in the nuclear fraction but also in the mitochondrial fraction. In both paradigms, no significant change of GAPDH levels occurred in the microsomal and cytosolic fractions. Moreover, pretreatment with GAPDH antisense oligonucleotide or classic apoptotic inhibitors clearly suppressed the accumulation of GAPDH protein in these subcellular loci. This discrete nuclear localization of GAPDH during apoptosis was supported further by immunoelectron microscopy. Quantitative assessment of GAPDH immunogold labeling revealed that a approximately 5-fold increase in the intensity of gold particles was observed within the nucleus of apoptotic cells. Thus, the current results raise the possibility that neuronal apoptosis may be triggered by GAPDH accumulation in the nucleus, resulting in perturbation of nuclear function and ultimate cell death.


Subject(s)
Apoptosis , Cell Nucleus/enzymology , Cerebellum/enzymology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Neurons/enzymology , Animals , Antibodies, Monoclonal/chemistry , Apoptosis/immunology , Cells, Cultured , Cerebellum/cytology , Glyceraldehyde-3-Phosphate Dehydrogenases/biosynthesis , Glyceraldehyde-3-Phosphate Dehydrogenases/immunology , Immunohistochemistry , Mice , Mice, Inbred BALB C , Microscopy, Immunoelectron , Rats , Rats, Sprague-Dawley , Subcellular Fractions/enzymology
14.
Bioorg Med Chem ; 6(4): 441-63, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9597188

ABSTRACT

A series of (4S)-4-[(2S)-benzyl-3-mercaptopropionylamino]-4-(N-phenylcarbamoyl )-butyric acids has been identified as potent systemically active enkephalinase inhibitors. Structure-activity relationships (SAR) are discussed. Further chemical modification of the inhibitors was carried out in order to identify the inhibitors which are orally active in an animal model. Compounds of particular interest are the prodrug-like analogues, including 5b (ONO-9902). Their analgesic effects after oral administration were evaluated.


Subject(s)
Analgesics/chemical synthesis , Butyrates/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Neprilysin/antagonists & inhibitors , Prodrugs/chemical synthesis , Administration, Oral , Analgesics/administration & dosage , Analgesics/pharmacokinetics , Analgesics/pharmacology , Analgesics, Non-Narcotic/administration & dosage , Analgesics, Non-Narcotic/chemical synthesis , Analgesics, Non-Narcotic/pharmacokinetics , Analgesics, Non-Narcotic/pharmacology , Animals , Area Under Curve , Biological Availability , Butyrates/administration & dosage , Butyrates/pharmacokinetics , Butyrates/pharmacology , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Dogs , Enkephalins , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Magnetic Resonance Spectroscopy , Male , Pain Measurement , Prodrugs/administration & dosage , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship
15.
Neurosci Lett ; 214(2-3): 151-4, 1996 Aug 23.
Article in English | MEDLINE | ID: mdl-8878106

ABSTRACT

We have reported that the antidementia drug tetrahydroaminoacridine (THA; 30 microM) is neuroprotective and neurotrophic and selectively increases m3-muscarinic acetylcholine receptor (mAChR) mRNA levels in differentiating cerebellar granule cells. Here, we examined whether novel prolyl endopeptidase inhibitor ONO-1603, a potential antidementia drug, induces similar effects in these cerebellar neurons. Supplement of ONO-1603 (0.03 microM) to cultures grown in 15 mM KCl-containing media was found to markedly promote neuronal survival and neurite outgrowth and enhance [3H]N-methylscopolamine binding to mAChRs. Moreover, ONO-1603 increased the level of m3-mAChR mRNA and stimulated mAChR-mediated phosphoinositide turnover. The common actions of ONO-1603 and THA suggest that these properties could be related to their putative antidementia activities and that this model system may be used to screen for drugs effective in the treatment for Alzheimer's disease.


Subject(s)
Brain Edema/drug therapy , Cerebellum/cytology , Muscarinic Agonists/pharmacology , Neuroprotective Agents/pharmacology , Pyrrolidines/pharmacology , RNA, Messenger/biosynthesis , Receptors, Muscarinic/biosynthesis , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Animals , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Cerebellum/drug effects , Cholinesterase Inhibitors/pharmacology , Neurons/drug effects , Phosphatidylinositols/metabolism , Physostigmine/pharmacology , Prolyl Oligopeptidases , Rats , Rats, Sprague-Dawley , Receptors, Muscarinic/metabolism
16.
J Pharmacol Exp Ther ; 278(1): 447-54, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8764381

ABSTRACT

We recently reported that the age-induced apoptotic death of cultured cerebellar neurons is correlated with an increased expression of a particulate-bound 38-kDa protein that we identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To determine whether this phenomenon of GAPDH overexpression occurs in other cell types, we selected primary cultures of cerebrocortical cells for testing, because under normal culture conditions, cortical neurons die progressively after 15 days in vitro. As with cerebellar neurons, this age-induced neuronal death involves ultrastructural changes and internucleosomal DNA fragmentation characteristic of apoptosis and is effectively prevented by actinomycin-D and cycloheximide. Moreover, a GAPDH antisense oligodeoxyribonucleotide arrested this cortical neuronal death for about 4 to 5 days and thus was more effective than cycloheximide. By contrast, its corresponding sense oligonucleotide had no effect. Additionally, the age-induced apoptosis of cortical neuronal cultures is effectively protected by aurintricarboxylic acid and tetrahy-droaminoacridine (an antidementia drug). Before cell death, GAPDH mRNA levels increased by about 2-fold and the increase was blocked by the above-mentioned neuroprotective agents and the GAPDH antisense, but not sense, oligonucleotide. The effects of antisense oligonucleotide are more robust in the present case than those found with cerebellar neurons, and they indicate a significant, though at present not defined, role of GAPDH in the apoptotic process occurring in these two types of neurons.


Subject(s)
Aging/metabolism , Apoptosis/drug effects , Cerebral Cortex/enzymology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Oligonucleotides, Antisense/pharmacology , Animals , Cells, Cultured , Rats
17.
J Neurochem ; 66(3): 928-35, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8769851

ABSTRACT

Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.


Subject(s)
Aging/physiology , Cerebellum/physiology , Glyceraldehyde-3-Phosphate Dehydrogenases/physiology , Neurons/physiology , Amino Acid Sequence , Animals , Apoptosis/physiology , Base Sequence , Cells, Cultured , Cellular Senescence , Cerebellum/cytology , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Molecular Sequence Data , N-Methylaspartate/pharmacology , Neuroprotective Agents/pharmacology , Oligonucleotide Probes/genetics , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley
18.
Prostaglandins Leukot Essent Fatty Acids ; 49(4): 795-804, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8259376

ABSTRACT

The effects of OP-41483.alpha-CD, 5(E)-6,9-deoxa-6,9 alpha-methylene-15-cyclopentyl-16,17,18,19,20-pentanor prostacyclin (PGI2).alpha-cyclodextrin clathrate, on platelet function and experimental thrombosis were studied. In human platelets, OP-41483 inhibited aggregation induced by adenosine diphosphate (ADP) or collagen, promoted disaggregation, and elevated cyclic adenosine monophosphate (cAMP) levels in vitro at the same order of concentrations. The equipotent antiaggregatory activity of OP-41483 to human platelets was observed in monkey platelets in vitro. Furthermore, intravenous administration of OP-41483 to monkeys, unlike PGI2, showed the antiaggregatory effect on platelets but with less effect on blood pressure, suggesting that a differential sensitivity to OP-41483 between platelet function and vascular tone exists in monkeys. In rabbits, OP-41483.alpha-CD attenuated platelet aggregation induced by ADP, collagen and platelet activating factor (PAF), decreased circulating platelet aggregates, and inhibited platelet adhesiveness to de-endothelialized blood vessels. These results suggest that the anti-thrombotic effects of OP-41483 are associated with its potent antiplatelet activities mainly because of the elevation of cAMP levels in the platelets. The potent anti-thrombotic and less hypotensive effects of this compound may be useful for various thrombotic disorders.


Subject(s)
Blood Platelets/drug effects , Cyclodextrins/pharmacology , Epoprostenol/analogs & derivatives , Fibrinolytic Agents/pharmacology , Platelet Activating Factor , Platelet Adhesiveness/drug effects , Platelet Aggregation Inhibitors/pharmacology , alpha-Cyclodextrins , Adenosine Diphosphate/pharmacology , Animals , Arachidonic Acid/pharmacology , Blood Coagulation Factors/pharmacology , Blood Pressure/drug effects , Collagen/pharmacology , Dogs , Epoprostenol/pharmacology , Female , Humans , Macaca , Male , Platelet Aggregation/drug effects , Platelet Count/drug effects , Rabbits , Rats , Rats, Wistar , Species Specificity , Thrombocytopenia/prevention & control
19.
Life Sci ; 51(4): 285-93, 1992.
Article in English | MEDLINE | ID: mdl-1625520

ABSTRACT

We examined the effects of arginine-vasopressin (AVP) C-terminal fragment 4-9, which facilitates learning and memory, on the extracellular acetylcholine (ACh) release in hippocampus of freely-moving rats using the microdialysis technique. Following administration of AVP4-9, p-Glu-Asn-Cys[Cys]-Pro-Arg-Gly-NH2, through the dialysis probe into the hippocampus, ACh levels in dialysates from the hippocampus increased markedly in dose and time dependent manner at 2-2.5 and 2.5-3 hr. AVP1-9, the parent peptide, has a similar enhancing effect on ACh release as AVP4-9. Stimulated ACh release by AVP4-9 was significantly inhibited by V1-selective receptor antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)-tyrosine]AVP), but not by V2-selective antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-D-Ile, 4-Ile]AVP). From these observations, it is demonstrated that AVP4-9 stimulates the ACh release in rat hippocampus via mediating V1-like vasopressin receptors.


Subject(s)
Acetylcholine/metabolism , Arginine Vasopressin/pharmacology , Hippocampus/drug effects , Peptide Fragments/pharmacology , Amino Acid Sequence , Analysis of Variance , Animals , Arginine Vasopressin/antagonists & inhibitors , Dialysis/methods , Hippocampus/metabolism , Locomotion , Male , Molecular Sequence Data , Peptide Fragments/antagonists & inhibitors , Rats , Rats, Inbred Strains
20.
J Med Chem ; 31(1): 84-91, 1988 Jan.
Article in English | MEDLINE | ID: mdl-3336036

ABSTRACT

(p-Amylcinnamoyl)anthranilic acid (3a) had moderate antagonist activities against LTD4-induced smooth muscle contraction on guinea pig ileum and LTC4-induced bronchoconstriction in anesthetized guinea pigs. Modifications were made in the hydrophobic part (cinnamoyl moiety) and the hydrophilic part (anthranilate moiety) of 3a. A series of 8-(benzoylamino)-2-tetrazol-5-yl-1,4-benzodioxans and 8-(benzoylamino)-2-tetrazol-5-yl-4-oxo-4H-1-benzopyrans were revealed to be potent antagonists of leukotrienes C4 and D4. Among both series, ONO-RS-347 (18k) and ONO-RS-411 (19h) were the most potent and orally active antagonists, respectively. Structure-activity relationships are discussed.


Subject(s)
Bronchodilator Agents/chemical synthesis , Cinnamates/chemical synthesis , Muscle Contraction/drug effects , SRS-A/antagonists & inhibitors , ortho-Aminobenzoates/chemical synthesis , Animals , Cinnamates/pharmacology , Guinea Pigs , Ileum/physiology , In Vitro Techniques , Magnetic Resonance Spectroscopy , Mass Spectrometry , SRS-A/pharmacology , Structure-Activity Relationship , ortho-Aminobenzoates/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...