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1.
Heart ; 90(4): 358-60, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15020494

ABSTRACT

Elective percutaneous coronary intervention fulfils many of the criteria needed of a clinical model of ischaemic preconditioning. But is this really a reflection of the laboratory phenomenon of ischaemic preconditioning?


Subject(s)
Balloon Occlusion/methods , Ischemic Preconditioning, Myocardial/methods , Adaptation, Physiological/physiology , Humans , Potassium Channels/physiology
2.
Infect Immun ; 67(8): 4243-50, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10417198

ABSTRACT

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.


Subject(s)
Antigens, Bacterial/immunology , Bacterial Vaccines/immunology , Luminescent Proteins/immunology , Mycobacterium avium/immunology , Recombinant Fusion Proteins/immunology , Vaccines, DNA/immunology , 3T3 Cells , Animals , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Cytokines/genetics , Female , Green Fluorescent Proteins , Humans , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Plasmids , RNA, Messenger/analysis , Reproducibility of Results , T-Lymphocyte Subsets/immunology , Tuberculosis/prevention & control , Vaccination
4.
Oncol Rep ; 4(6): 1373-81, 1997.
Article in English | MEDLINE | ID: mdl-21590256

ABSTRACT

The ras family of oncogenes are the most frequently activated group of dominant transforming genes in both human and experimental cancers. The ras family of genes encode highly similar proteins with molecular weights of 21 kDa which are thought to play a key role in signal transduction. Activation in vivo by point mutations results in the ras p21 protein being maintained in the activated form and stimulating cellular proliferation autonomously. Point mutations at codon 12 of K-ras have been observed in >75% of cases of adenocarcinomas of the exocrine pancreas. The type and frequency of K-ras gene mutations in pancreatic cancer cell lines and in bile samples from patients with cytologically-proven biliary tract malignancies and from patients with non-malignant disorders of the biliary tract were determined. Codons 12, 13 and 61 of the K-ras gene were analysed by using restriction fragment length polymorphisms created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. A mutation of codon 12 of K-ras was detected in 10 of 13 (77%) human pancreatic cancer cell lines. The amino-acid substitutions were glycine to aspartate (5 samples), arginine (2), valine (2) and cysteine (1). No mutations were found at codons 13 or 61. A mutation at codon 12 of K-ras was detected in 9 of 18 (50%) of bile samples analysed. Eleven bile samples had positive cytology for malignancy of pancreaticobiliary origin, and 4 (36%) of these had a codon 12 mutation. Mutations were detected in 5 of the 7 (71%) cytologically-negative bile samples, although malignancy was subsequently diagnosed in 2 of these patients on further histology, and was suspected in 3 other cases on clinical and radiological criteria. This method provides a rapid determination of K-ras gene mutations in bile samples for patients with pancreatic and biliary tract diseases, which may be useful when considering future therapy directed at inhibition of activated ras-induced signal transduction pathways.

5.
Mol Pharmacol ; 49(6): 1033-41, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8649341

ABSTRACT

The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors. Here, we report that Leu- and Met-enkephalin and their des-tyrosyl derivatives potently and selectively inhibited high affinity L-proline uptake in rat hippocampal synaptosomes and in PROT-transfected HeLa cells. High concentrations of the opiate receptor antagonist naltrexone did not block the inhibitory actions of these peptides, arguing against an involvement of opioid receptors. Des-tyrosyl-Leu-enkephalin elevated the apparent K(m) of L-proline transport in transfected HeLa cells without altering the V(max). PROT-transfected HeLa cells did not accumulate [3H]Leu-enkephalin above background levels, demonstrating that enkephalins are not substrates for PROT. These findings indicate that enkephalins competitively inhibit mammalian brain PROT through a direct interaction with the transporter protein at or near the L-proline binding site. The high potency and specificity of des-tyrosyl-Leu-enkephalin make this compound a useful tool for elucidating the structure-function properties and physiological role(s) of PROT.


Subject(s)
Amino Acid Transport Systems, Neutral , Brain/drug effects , Enkephalins/pharmacology , Membrane Transport Modulators , Membrane Transport Proteins/antagonists & inhibitors , Animals , HeLa Cells , Humans , Male , Naltrexone/pharmacology , Proline/metabolism , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship
6.
Am J Physiol ; 270(1 Pt 1): C67-75, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8772431

ABSTRACT

The extracellular concentration of glutamate and other related excitatory amino acids (EAA) is regulated by the action of transporter proteins located on either presynaptic terminals or adjacent astroglial processes. Recent molecular advances have led to the cloning of three separate cDNAs encoding for Na(+)-dependent glutamate transporters; two are thought to be primarily glial in origin (GLAST and GLT-1) and the third (EAAC1) is localized to neurons in the brain and other nonneural tissues. An EAAC1 cDNA was initially cloned from rabbit small intestine (13). In this study, we report isolation and characterization of the homologous clone from rat brain. Northern blot hybridization revealed high levels of EAAC1 mRNA in rat brain and kidney and low levels in heart, lung, and skeletal muscle. Transient expression of EAAC1 in HeLa cells resulted in an increase in Na(+)-dependent high-affinity L-[3H]glutamate and D-[3H]aspartate transport. The pharmacological profile of EAAC1 was very similar to that reported for the rabbit and human EAAC1 homologues. Transport activity was potently inhibited by D- and L-threo-beta-hydroxyaspartate and L-trans-pyrrolodine-2,4-dicarboxylate. Dihydrokainate and L-alpha-aminoadipate did not inhibit transport at concentrations below 1 mM. Oligonucleotide cDNA probes (45-mer) were constructed and labeled with 35S-ATP for film- and emulsion-based in situ hybridization of rat brain. EAAC1 mRNA had the highest density in the cerebellar granule cell layer, hippocampus, superior colliculus, and neocortex. Sections that were emulsion-dipped and counterstained with cresyl violet revealed EAAC1 labeling localized exclusively over neuronal cell bodies, including some nonglutamatergic neurons such as spinal cord ventral horn cells.


Subject(s)
Amino Acid Transport System X-AG , Brain/metabolism , Carrier Proteins/metabolism , Neurons/metabolism , Symporters , Amino Acid Sequence , Animals , Brain/cytology , Carrier Proteins/genetics , Cloning, Molecular , Excitatory Amino Acid Transporter 1 , Excitatory Amino Acid Transporter 3 , Glutamate Plasma Membrane Transport Proteins , HeLa Cells/metabolism , Hippocampus/metabolism , Humans , Male , Molecular Sequence Data , Oligonucleotide Probes/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tissue Distribution
7.
Mol Pharmacol ; 48(2): 219-29, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7651355

ABSTRACT

L-Proline fulfills several of the classic criteria used to identify amino acid neurotransmitters, including the presence of a high affinity, Na(+)- (and Cl-)-dependent synaptosomal transport process and the Ca(2+)-dependent release of exogenously loaded radiolabeled L-proline from brain slices and synaptosomes after K(+)-induced depolarization. However, studies to define the role of L-proline in discrete pathways in the mammalian brain have been precluded by the inability to block its biosynthesis or high affinity transport in nervous tissue. We report the molecular cloning, functional expression, and chromosomal localization of a human brain-specific high affinity L-proline transporter (hPROT). The pharmacological specificity, kinetic properties, and ionic requirements of hPROT clearly distinguish this carrier from the other Na(+)-dependent plasma membrane carriers that transport L-proline. Multiple tissue Northern blot analysis revealed a prominent approximately 4-kb mRNA transcript in human brain tissue, whereas no specific hybridizing species were detected in peripheral tissue. An antipeptide antiserum directed against the carboxy-terminus of the predicted hPROT protein identified a single, broad immunoreactive protein of 68 kDa on immunoblots of synaptosomal membranes from various human brain regions. In contrast, no specific labeling was detected on immunoblots of membranes from human liver, kidney, or heart. A differential distribution of hPROT mRNA and protein was observed in the human corpus striatum, consistent with the hypothesis that the hPROT protein is synthesized in neuronal cell bodies in an extrastriatal location and axonally transported to the corpus striatum. These findings warrant the consideration of a synaptic regulatory role for this transporter and its presumed natural substrate, L-proline, in the mammalian central nervous system.


Subject(s)
Amino Acid Transport Systems, Neutral , Brain/metabolism , Chromosome Mapping , Membrane Transport Proteins/genetics , Adult , Amino Acid Sequence , Animals , Base Sequence , Biological Transport , Cloning, Molecular , Corpus Striatum/metabolism , DNA, Complementary , Humans , Membrane Transport Proteins/chemistry , Mice , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism
8.
J Biol Chem ; 270(26): 15755-61, 1995 Jun 30.
Article in English | MEDLINE | ID: mdl-7797577

ABSTRACT

The expression of a high affinity Na(+)- (and Cl-) dependent L-proline transporter (PROT) in subpopulations of putative glutamatergic pathways in rat brain raises the possibility of a specific physiological role(s) for this carrier in excitatory neurotransmission (Fremeau, R. T., Jr., Caron, M. G., and Blakely, R. D. (1992) Neuron 8, 915-926). However, the biochemical properties and regional, cellular, and subcellular distribution of the PROT protein have yet to be elucidated. Here, we document the brain-specific expression and neuronal localization of rat PROT mRNA. We also report the first identification and partial biochemical characterization of the mammalian brain PROT protein. An affinity-purified antipeptide antibody was produced that specifically recognized a single 68-kDa PROT protein on immunoblots of rat and human brain tissues. Deglycosylation of rat hippocampal membranes with peptide-N-glycosidase F reduced the apparent molecular mass of the native PROT protein from 68 to 53 kDa, the size of the primary PROT translation product determined by in vitro translation of the rat PROT cDNA in the absence of microsomes. Subcellular fractionation studies demonstrated that the PROT protein was enriched in synaptic plasma membranes but absent from postsynaptic densities. A differential distribution of PROT mRNA and protein was observed in rat striatum, suggesting that the transporter protein is synthesized in neuronal cell bodies in the cortex and exported to axon terminals in the caudate putamen. These findings warrant the consideration of a novel presynaptic regulatory role for this transporter in excitatory synaptic transmission.


Subject(s)
Amino Acid Transport Systems, Neutral , Brain Chemistry , Membrane Transport Proteins/analysis , Nerve Tissue Proteins/analysis , Neurons/chemistry , RNA, Messenger/analysis , Synaptic Membranes/chemistry , Amino Acid Sequence , Animals , Glycoproteins/analysis , Male , Membrane Transport Proteins/genetics , Membrane Transport Proteins/physiology , Molecular Sequence Data , Protein Biosynthesis , Rats , Rats, Sprague-Dawley , Transcription, Genetic
10.
J Virol Methods ; 23(2): 157-67, 1989 Feb.
Article in English | MEDLINE | ID: mdl-2542350

ABSTRACT

We have developed a murine monoclonal antibody reactive with a major immediate early 72,000 dalton protein of human cytomegalovirus and utilized this reagent in a rapid virus titration and microneutralization assay. Because of the early expression of this virus encoded protein, both assays could be accomplished within 16 h following virus inoculation. In addition, both assays resulted in considerable savings of reagents because the assays were carried out in 96-well microtiter plates. These assays should prove useful in the preparation and study of neutralizing antibodies directed against human cytomegalovirus.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/analysis , Cytomegalovirus/immunology , Immediate-Early Proteins , Antigens, Viral/immunology , Cells, Cultured , Fibroblasts , Humans , Immunoblotting , Neutralization Tests , Nuclear Proteins/immunology
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