Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.2.42 (DTA)
 1,693 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A genetically engineered gene fusion was constructed which encoded a nontoxic derivative of the A fragment of diphtheria toxin joined to the C180 peptide of the S1 subunit of pertussis toxin. The product of this gene fusion, termed the DTA-C180 protein, was purified from the periplasm of Escherichia coli to approximately 80% purity. The DTA-C180 protein possessed an apparent molecular weight of 43,000 by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DTA-C180 protein was cleaved into two tryptic peptides, which migrated with apparent molecular weights of approximately 22,000. One tryptic peptide reacted with diphtheria antitoxin, while the other tryptic peptide reacted with anti-C180 peptide immunoglobulin G. The DTA-C180 protein did not inhibit protein synthesis or stimulate clustering morphology in Chinese hamster ovary cells. The DTA-C180 protein elicited an immune response, in guinea pigs, against both the DTA and C180 peptide components of the fusion protein, with alum being a more efficient adjuvant than Freund's adjuvant for eliciting neutralization titers. Neutralization titers elicited by DTA-C180 protein were weaker than those elicited by diphtheria toxoid and pertussis toxin 9K/129G, a genetically engineered double mutant of pertussis toxin. Three doses of DTA-C180 protein yielded a neutralization titer of 1/750 against pertussis toxin in Chinese hamster ovary cells and a neutralization titer of 1/50 against diphtheria toxin in Vero cells. This is the first report of a protein derived from a recombinant S1 subunit that elicits a neutralizing titer against pertussis toxin.
Infect Immun 1992 Dec
PMID:Construction of a diphtheria toxin A fragment-C180 peptide fusion protein which elicits a neutralizing antibody response against diphtheria toxin and pertussis toxin. 145 39

Forty-one patients with probable dementia of the Alzheimer type (DAT) have been studied by computerized EEg (C-EEG) and single photon emission tomography (SPECT) using 123-IMP. Four groups have been distinguished according to SPECT hypoperfusion topography: frontal, temporo-parietal, fronto-parietal and fronto-temporo-parietal. C-EEG parameters were much more disturbed in the 2 latter than in the 2 former groups. Thus a normal C-EEG could indicate a dominant frontal or temporo-parietal form of DAT. Therefore some bias due to duration or rate of disease progression cannot be excluded. So far the important finding is that EEG changes are diffuse and do not reflect the topography of SPECT hypoperfusion questioning the value of EEG topographical analysis in DTA.
Neurophysiol Clin 1991 Dec
PMID:[Comparison of SPECT and quantified EEG features in Alzheimer's type dementia]. 180 97

By combining dioleoylphosphatidylethanolamine (DOPE) with oleic acid (OA), palmitoylhomocysteine (PHC) or dipalmitoylsuccinylglycerol (DPSG) we have prepared pH-sensitive liposomes with different acid sensitivities. DOPE/OA liposomes are the most acid sensitive, while DOPE/DPSG liposomes are the least acid sensitive. Incubation of DOPE/OA liposomes with mouse L929 cells reduces the pH-sensitivity of these liposomes by altering the lipid composition. Using diphtheria toxin fragment A as a marker for cytoplasmic delivery, we find that the delivery kinetics of pH-sensitive immunoliposomes closely correlates with the modified acid sensitivities of the liposomes. Immunoliposomes encounter pH 6-6.2 with a t1/2 of 5-15 min after internalization. By contrast, acidification of the endosomes to pH 5.0 takes longer (t1/2 approximately 25 min). We also used a whole cell null point technique (Yamishiro and Maxfield (1987) J. Cell Biol. 105, 2713-2721) to directly determine the average pH encountered by the endocytosed immunoliposomes. We find that acidification determined by the null point method proceeds less rapidly than that estimated from DTA delivery data. This is likely due to the fact that the measured DTA delivery is done by those liposomes which first arrive at the endosomes with sufficient acidity. Our data suggests that DOPE/PHC immunoliposomes deliver at the early endosome while DOPE/DPSG immunoliposomes deliver at the late endosomes. The DOPE/OA immunoliposomes, with the altered composition and acid sensitivity, deliver with a kinetics intermediate between the other two immunoliposomes. Thus, pH-sensitive liposomes represent useful probes for studying the kinetics of endosome acidification.
Biochim Biophys Acta 1989 Dec 11
PMID:Immunoliposomes with different acid sensitivities as probes for the cellular endocytic pathway. 259 86

Previous studies have suggested that tyrosine-65 (Tyr-65) of diphtheria toxin (DT) is located at the active site. To investigate the role of Tyr-65 in NAD binding and the ADP-ribosylation of elongation factor-2 (EF-2), we changed this residue to alanine and phenylalanine by site-directed mutagenesis of a synthetic gene encoding the catalytic fragment of DT (DTA). The alanine mutant was greatly diminished in ADP-ribosylation activity (350-fold) and NAD-glycohydrolase activity (88-fold), whereas the phenylalanine mutant was reduced in these activities only slightly. Dissociation constants (Kd) for NAD binding were 15 microM for wild-type DTA, 26 microM for the phenylalanine mutant, and greater than 800 microM NAD for the alanine mutant. However, both mutant enzymes were found to bind adenosine with nearly equal affinity as wild-type DTA. These results support a model of ADP-ribosylation in which the phenolic ring of Tyr-65 interacts with the nicotinamide ring of NAD, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism.
Biochemistry 1994 Dec 27
PMID:Active-site mutations of diphtheria toxin: role of tyrosine-65 in NAD binding and ADP-ribosylation. 780 11

The bacteriophage T3 promoter can be selectively transcribed by the corresponding RNA polymerase in eukaryotic cells. A toxic gene can in principle be linked to this promoter in a "dormant" and innocuous transgene in a transgenic animal. In this scheme, the activating strain expresses the RNA polymerase. When expression of the gene is needed in the progeny, the 2 lines are crossed. However, when a single molecule is sufficient to kill the cell--as with the diphtheria toxin--transcriptional "leakage" from the promoter may not be tolerated by the cell, even when extremely weak. Therefore, prior to more elaborate studies, diphtheria toxin, as a prototype of a gene toxic to the organism, has been linked to the bacteriophage T3 promoter in a T3-E-DTA construct. The T3-E-DTA plasmid has been transiently transfected into human embryonic kidney derived cells together with a lacZ plasmid. By co-transfection, the T3-E-DTA cells can be readily identified as lacZ positive, and their fate followed by the production of beta-galactosidase at the single cell or overall population level. In spite of the extreme toxicity of the toxin, the cells tolerate the presence of the T3-E-DTA construct, and are only killed--with a high efficiency--when the T3 RNA polymerase is present. Transactivation is usually restricted to the auxiliary factors of transcription. With this study, the promoter and the polymerase are revealed as potential and efficient inducible and activating elements of a very simple binary system.
C R Acad Sci III 1996 Dec
PMID:A bacteriophage T3 promoter can be linked to a lethal gene without detectable toxicity for eukaryotic cells. Interest for inducible transgenes. 909 Nov 77

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.
J Clin Invest 1997 Dec 01
PMID:Troglitazone action is independent of adipose tissue. 938 57

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20-24 residue peptides of FhuB by determining the activity of beta-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA...G (in FhuB DTA ...G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.
Mol Microbiol 1997 Dec
PMID:ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping. 942 46

Anthrax lethal toxin is a mixture of protective antigen (PA, 735 AA) and lethal factor (LF, 776 AA). Earlier studies have shown that 254 residues of lethal factor are sufficient for PA binding to cause internalization (Arora N and Leppla SH, J Biol Chem 268: 3334-3342, 1993). The present study was undertaken to determine residues which are important for binding of LF to PA. LF modification with diethyl pyrocarbonate (DEPC, modifies histidine residue primarily) results in the loss of binding and toxicity in mammalian cells. There are nine histidine residues in the binding domain. To locate the important residue(s), site-directed mutagenesis of these histidines were performed by recombinant methods. Replacement of His42 with Gly42 destablizes the protein and hence it could not be purified. His35 when mutagenized to Gly35 (mLF-DTA) diminishes the toxicity by 20 fold. Time dependent studies show that binding of mLF-DTA was reduced at shorter incubations and longer incubations taper off this difference. Gel shift assay suggested 8-10% less binding of mLF-DTA as compared to LF-DTA. In conclusion His35 is important for binding and His42 is critical and confers proper conformation for LF binding to PA.
Mol Cell Biochem 1997 Dec
PMID:Site directed mutagenesis of histidine residues in anthrax toxin lethal factor binding domain reduces toxicity. 945 Jun 39

Glass-ceramic powders with the oxide batch formulation 50wt%SiO2, 20wt%P2O5 and 30wt%CaO were prepared by the sol-gel process using tetraethoxysilane, calcium nitrate and phosphoric acid as raw materials. The obtained gels and powders were characterized by TG-DTA thermal analysis, FTIR analyses and XRD measurements. The results indicated that (1) heating to more than 600 degrees C brought about elimination of -OC2H5 side chains from gels, and formation of a SiO2-based glass network, (2) crystallization such as Ca3(PO4)2 notably occurred in the SiO2-based glass matrix when heated to more than 900 degrees C, (3) -OH groups existed on SiO2-based glass-ceramic surfaces in the temperature region less than 900 degrees C, and (4) the melting point of this composition was more than 1400 degrees C. This study suggests that sol-gel-derived SiO2-based glass-ceramic powder containing P and Ca might be useful as bioactive implant materials.
Dent Mater J 1997 Dec
PMID:Characterization of 50wt%SiO2-20wt%P2O5-30wt%CaO glass-ceramic powders prepared by the sol-gel process. 955 50

Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 microM of either linoleic acid (LA, 18:2n-6), gamma-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 microM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.
Biosci Biotechnol Biochem 2000 Dec
PMID:Modulation of cholesterol concentration in Caco-2 cells by incubation with different n-6 fatty acids. 1121 Jan 14


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