Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G985. The same assay consistently revealed A985 in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G985 mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.
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PMID:Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene, and expression of inactive mutant enzyme protein in E. coli. 190 18

To facilitate molecular analyses of a previously uncharacterized gene involved in alanine synthesis, attempts were made to clone the wild-type allele of this gene, alaA, with a mini-Mu plasmid element used for in vivo cloning. Seventy-six independent Ala+ plasmids were isolated and characterized. Physiological, enzymological, and restriction endonuclease analyses indicated that three different genes, none of them alaA, were cloned. These genes were avtA+, which encodes the alanine-valine transaminase (transaminase C); tyrB+, which encodes the tyrosine-repressible transaminase (transaminase D); and a previously undescribed gene, called alaB, which encodes an alanine-glutamate transaminase.
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PMID:Cloning of genes that suppress an Escherichia coli K-12 alanine auxotroph when present in multicopy plasmids. 289 Jun 23

Purified Anabaena variabilis chromosomal DNA was partially digested with restriction endonuclease Sau3A and ligated into the BamHI site of plasmid pBR322. Escherichia coli 342-167, a mutant with a decreased level of phosphoenolpyruvate carboxylase (PEPCase) activity was transformed with plasmids from the A. variabilis genomic library. A transformant that grew on minimal media in the absence of glutamate was isolated and its plasmid, pTRH1, was shown to encode the A. variabilis PEPCase. E. coli HB101 cells transformed with plasmid pTRH1 have approx. 50 times the normal amount of PEPCase activity and also overproduce a protein with the apparent Mr (99,000) of the A. variabilis PEPCase.
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PMID:Molecular cloning of the phosphoenolpyruvate carboxylase gene of Anabaena variabilis. 309 19

The gene for the H-2K class I antigen of the bm1 variant was cloned and analyzed at the DNA level and compared with the previously cloned parent B6/Kh gene. Sequence determination and comparative restriction endonuclease studies indicate that Kbm1 is derived from the Kb gene. Seven nucleotide changes within a 13-nucleotide stretch distinguish the mutant from the parent gene and result in amino acid differences at positions 152, 155, and 156 in the antigen. The data confirm previously reported changes at amino acid positions 155 and 156 (arginine to tyrosine and leucine to tyrosine, respectively) and extend the altered region to include two nucleotides encoding a glutamate to alanine substitution at amino acid 152, a change not detected by the protein studies because of limitations of the methods used. The DNA sequence encoding this region of the Kbm1 glycoprotein is identical to the DNA sequence of at least one other known class I gene in the mouse, a finding consistent with the hypothesis that the mutation was not a random event but may be the result of a block transfer of information by a copy mechanism analogous to gene conversion. As the sequence analysis of the coding region for the first 273 amino acid residues shows identity between parent and mutant except for the seven nucleotide changes, all variant-parent functional differences must depend only on the cluster of three amino acid differences in the second domain of the Kb glycoprotein.
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PMID:Comparison of the cloned H-2Kbm1 variant gene with the H-2Kb gene shows a cluster of seven nucleotide differences. 630 Aug 87

A dramatic loss of glutamate transport has been observed in sporadic amyotrophic lateral sclerosis and has been postulated to contribute to the disease. Experimentally, this hypothesis was corroborated by mimicking the chronic loss of glutamate transport in postnatal rat spinal cord organotypic cultures through the use of glutamate transport inhibitors. This system is characterized by a relatively selective slow loss of ventral horn motor neurons resulting from glutamate transport inhibition. In this study, spinal cord organotypic cultures were used to test various drugs to evaluate their neuroprotective properties against this slow glutamate-mediated neurotoxicity The most potent neuroprotectants were drugs that altered glutamate neurotransmission, including non-NMDA receptor antagonists (GYKI-52466, PD144216, and PD13997) and drugs that could block presynaptic release or synthesis (riluzole and gabapentin). In addition, some antioxidants (U83836E and N-t-butyl-alpha-phenylnitrone) and inhibitors of nitric oxide synthesis (NG-monomethyl-L-arginine acetate) were modestly neuroprotective. The calcium endonuclease inhibitor aurintricarboxylic acid and the calcium release inhibitor dantrolene also provided partial motor neuron protection. However, several antioxidants and calcium channel antagonists had no excitotoxic neuroprotectant activity. This system provides a preclinical screening method for the burgeoning number of drugs postulated for clinical trials in motor neuron disease and a model to evaluate the mechanisms of chronic glutamate toxicity.
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PMID:Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity. 761 20

All organisms express dedicated repair enzymes for counteracting the cytotoxic and mutagenic potential of apurinic/apyrimidinic (AP) lesions, which would otherwise pose a serious threat to genome integrity. We present the predicted three-dimensional structure of the major human AP site-specific DNA repair endonuclease, HAP1, and show that an aspartate/histidine pair, in conjunction with a metal ion-coordinating glutamate residue, are critical for catalyzing the multiple repair activities of HAP1. We suggest that this catalytic mechanism is conserved in certain reverse transcriptases, but is distinct from the two metal ion-mediated mechanism defined for other hydrolytic nucleases.
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PMID:Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1. 766 24

In this study, the endonuclease inhibitor aurintricarboxylic acid (ATA) was examined for its ability to attenuate both acute and delayed excitotoxicity mediated through NMDA and non-NMDA glutamate receptors. Ex vivo embryonic chick retina, a model system frequently used for studies of excitotoxicity, was exposed to either 100 microM NMDA or kainate (KA) +/- various concentrations of ATA for 60 min, then allowed to recover for 24 h. Lactate dehydrogenase release into the medium and histology were assessed as measures of delayed toxicity. ATA attenuated lactate dehydrogenase release due to NMDA or KA in a dose-dependent manner. Histology revealed that ATA decreased the number of pyknotic profiles in response to either glutamate agonist. The mechanism of ATA protection was addressed. ATA was found to block NMDA- but not KA-mediated 22Na+ influx and cyclic GMP formation. In membrane binding studies, ATA was relatively selective for displacement at the NMDA receptor. The IC50 values for displacement of [3H]CGS 19755, alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), or [3H]KA were 29.9 +/- 1.3, 313 +/- 46, and > 1,000 microM +/- SEM, respectively. ATA also fully attenuated NMDA-induced and partially attenuated KA-induced acute excitotoxicity as monitored histologically by tissue swelling and by the increase in GABA in the medium. Temporal studies of ATA efficacy indicated that ATA needed to be present during NMDA exposure to afford protection but, versus KA, was equally effective if administered immediately after KA exposure. Questions regarding the cellular penetration of ATA were raised because incubation with 100 microM ATA for 60 min had no effect on lactate formation or [3H]leucine incorporation into trichloroacetic acid-precipitable material, even though, in cell-free systems, ATA is a potent inhibitor of phosphofructokinase activity and protein synthesis. These studies demonstrate that ATA can protect against excitotoxicity mediated through NMDA or non-NMDA glutamate receptors. The mechanism of protection versus NMDA is through interruption of NMDA receptor interactions. ATA has no direct effect at the KA receptor; thus, its mechanism of protection versus KA is distinct from that versus NMDA and is, at present, unknown.
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PMID:Excitotoxicity at both NMDA and non-NMDA glutamate receptors is antagonized by aurintricarboxylic acid: evidence for differing mechanisms of action. 789 Nov 4

Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antisense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E. coli carrying the aadA gene construct, and transformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.
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PMID:Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease. 790 39

The non-specific endonuclease inhibitor, aurintricarboxylic acid (ATA), attenuated glutamate-induced destruction of cultured cortical neurons. In part, this protective effect likely reflected the ability of ATA to produce a slowly developing block of N-methyl-D-aspartate receptor-mediated inward whole cell current or increase in intracellular free Ca2+. However, ATA also attenuated a high K(+)-induced increase in intracellular free Ca2+ in the presence of D-amino-phosphonovalerate, suggesting that ATA may have a more general effect on Ca2+ homeostasis. In addition, ATA attenuated glutamate neurotoxicity even if added up to 2 hr after completion of glutamate exposure, a time when glutamate antagonists or lipid peroxidation inhibitors are no longer neuroprotective. Involvement of apoptosis in this excitotoxic death is unlikely, as Southern blotting of genomic DNA revealed no evidence of fragmentation, and death was not prevented by inhibitors of RNA or protein synthesis. Most likely, ATA interferes with some key downstream consequences of excitotoxic glutamate receptor overactivation.
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PMID:Delayed application of aurintricarboxylic acid reduces glutamate-induced cortical neuronal injury. 791 46

Activation of programmed cell death has recently been suggested to be involved in the delayed neuronal death of CA1 hippocampal neurons after global ischemia based on protection offered by protein synthesis inhibitors. Here, we studied the effects of transcriptional (actinomycin D) and translational (cycloheximide and anisomycin) inhibitors on glutamate-induced neuronal death in cerebellar granule cell cultures. The effects of aurintricarboxylic acid, an endonuclease inhibitor, were studied as well. No protection against glutamate toxicity could be observed with any of these inhibitors. We also analyzed the genomic DNA of glutamate-treated cells on agarose gel electrophoresis. No DNA degradation could be observed after glutamate exposure. We conclude that glutamate-induced neuronal death does not exhibit the features of apoptosis in cultured granule cells.
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PMID:Glutamate-induced neuronal death is not a programmed cell death in cerebellar culture. 809 39


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