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1.
Invest Ophthalmol Vis Sci ; 45(6): 1674-9, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15161825

ABSTRACT

PURPOSE: N-myristoyltransferase (NMT) is a ubiquitously distributed eukaryotic enzyme that catalyzes myristoylation of proteins. Very little is known about the process of myristoylation, particularly in the eye. In the present study, the distribution, expression, and correlation of NMT and its inhibitor (NMT inhibitor protein, NIP) in the bovine eye were investigated. METHODS: Whole bovine eyes were either homogenized or regionally dissected to determine the activity and localization of NMT and NIP. Dissected tissues were homogenized, and Western blot analysis was performed using polyclonal anti-NMT and anti-NIP antibodies. The NMT activity was assayed using cAMP-dependent protein kinase or pp60(src) derived peptide as a substrate. Fresh samples were then prepared for immunohistochemical analysis and probed with polyclonal anti-NMT and anti-NIP antibodies. RESULTS: The total bovine eye cytosolic fraction displayed both NMT and NIP expression. NMT was present in all the regions of the eye at various levels of expression. The highest expression of NMT was in the cornea, whereas NIP was present in the retina, optic nerve, sclera, and choroid only. NIP expression was the highest in the optic nerve, sclera, and retina. NMT activity was observed in the cornea, iris, and retina after DEAE-Sepharose CL-6B column chromatography. The inhibitory activity of crude homogenate on recombinant human NMT activity was found to be greater for optic nerve and choroid. Immunohistochemistry results displayed similar findings. CONCLUSIONS: The varied expression of NMT in different regions of the eye reveals a regulatory relationship of NMT with NIP. These findings indicate that NMT and NIP are present in various regions of the eye and will lead to further understanding of visual signaling in ocular cells.


Subject(s)
Acyltransferases/antagonists & inhibitors , Acyltransferases/metabolism , Enzyme Inhibitors/metabolism , Eye Proteins/metabolism , Eye/enzymology , Protein Processing, Post-Translational , Animals , Blotting, Western , Cattle , Electrophoresis, Polyacrylamide Gel , Immunoenzyme Techniques , Substrate Specificity
2.
Cancer Res ; 63(22): 7975-8, 2003 Nov 15.
Article in English | MEDLINE | ID: mdl-14633729

ABSTRACT

N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the NH(2) terminus of the glycine residue of a number of important proteins that have diverse biological functions and thus have been proposed as potential targets for chemotherapeutic drug design. Earlier, we demonstrated that NMT is more active in colonic epithelial neoplasms than in corresponding normal-appearing colonic tissue. Furthermore, an increased expression of NMT was also observed in gallbladder carcinoma. In the present study, we report a novel protein inhibitor of NMT. This protein caused a potent concentration-dependent inhibition of human NMT with half-maximal inhibition at 4.5 +/- 0.35 nM. This study will serve as a template for further investigations in the area of protein myristoylation.


Subject(s)
Acyltransferases/antagonists & inhibitors , Phosphopyruvate Hydratase/pharmacology , Acyltransferases/metabolism , Amino Acid Sequence , Dose-Response Relationship, Drug , Enzyme Stability , Humans , Molecular Sequence Data , Phosphopyruvate Hydratase/chemistry
3.
Nucleic Acids Res ; 31(5): 1502-13, 2003 Mar 01.
Article in English | MEDLINE | ID: mdl-12595559

ABSTRACT

The human SRC gene encodes pp60(c-src), a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes.


Subject(s)
DNA-Binding Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Pyrimidines/metabolism , Amino Acid Sequence , Binding Sites/genetics , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Chromatography, Affinity , Chromatography, Ion Exchange , Cross-Linking Reagents/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein K/genetics , Humans , Molecular Sequence Data , Mutation , Oligonucleotides/genetics , Oligonucleotides/metabolism , Promoter Regions, Genetic/genetics , Protein Binding/radiation effects , Proto-Oncogene Proteins pp60(c-src)/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transcription, Genetic , Tumor Cells, Cultured , Ultraviolet Rays
4.
Can J Physiol Pharmacol ; 80(1): 59-66, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11926171

ABSTRACT

In the present study, we investigated the activity and expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) and the effects of calpains in rat heart after ischemia and reperfusion. Immunohistochemical studies indicated that CaMPDE in normal heart is localized in myocardial cells. Rat ischemic heart showed a decrease in CaMPDE activity in the presence of Ca2+ and calmodulin; however, in ischemic-reperfusion tissue a progressive increase in Ca2+ and calmodulin-independent cyclic nucleotide phosphodiesterase (CaM-independent PDE) activity was observed. Perfusion of hearts with cell-permeable calpain inhibitor suppressed the increase of Ca2+ and CaM-independent PDE activity. Protein expression of CaMPDE was uneffected by hypoxic injury to rat myocardium. The purified heart CaMPDE was proteolyzed by calpains into a 45 kDa immunoreactive fragment in vitro. Based on these results, we propose that hypoxic injury to rat myocardium results in the generation of CaM-independent PDE by calpain mediated proteolysis, allowing the maintenance of cAMP concentrations within the physiological range.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Myocardial Reperfusion Injury/enzymology , Animals , Blotting, Western , Calpain/antagonists & inhibitors , Cattle , Cyclic AMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 1 , Immunohistochemistry , Ischemic Preconditioning, Myocardial , Male , Myocardium/enzymology , Rats , Rats, Sprague-Dawley
5.
Eur J Med Chem ; 37(1): 35-44, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11841873

ABSTRACT

A series of 1,4-bis(2-oxo-1-cycloalkylmethylene)benzenes 2a-c and 4 and a related acyclic analogue 6a were synthesised and converted to the corresponding Mannich bases 3a-c, 5 and 6b. Evaluation of these compounds against murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes revealed that the Mannich bases were more cytotoxic than the corresponding unsaturated ketones. 1,4-bis(3-Dimethylaminomethyl-2-oxo-1-cyclohexylmethylene)benzene dihydrochloride (3a) had lower IC(50) values than melphalan against the four cell lines and was 15 times more potent than this drug when examined against a panel of human tumours.


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Benzene Derivatives/chemical synthesis , Benzene Derivatives/pharmacology , Mannich Bases/chemical synthesis , Mannich Bases/pharmacology , Animals , Antineoplastic Agents/chemistry , Benzene Derivatives/chemistry , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Mannich Bases/chemistry , Mice , Structure-Activity Relationship , Tumor Cells, Cultured
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