Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four 25-nt long oligonucleotides containing dA and dT (D1, D2, D3, and D4) which are capable of forming parallel-stranded (ps) or antiparallel-stranded (aps) duplexes have been synthesized [Rippe, K., Ramsing, N. B., & Jovin, T. M. (1989) Biochemistry 28, 9536-9541]. In the present study, the OsO4-pyridine complex (Os,py), diethyl pyrocarbonate (DEPC), KMnO4, and the 1,10-phenanthroline-cuprous complex [(OP)2Cu+] were used to investigate the conformation-dependent reactivity of ps, aps, and single-stranded (ss) oligonucleotides. The products were analyzed by polyacrylamide gel electrophoresis with single-nucleotide resolution. The results confirm the duplex nature of the ps combinations of oligonucleotides and reveal structural differences in comparison with the aps molecules. Under conditions in which ss-DNA is substantially sensitive to Os,py, both the ps and aps duplexes are very unreactive. A similar result was observed with KMnO4 and DEPC, although with the latter reagent the modification pattern of the labeled strands D1* and D4* was slightly different for the parallel than for the antiparallel duplex. The (OP)2Cu+ complex efficiently cleaves the aps but not the ps duplex and shows a preference for TAT steps. We also tested the effect of monovalent and divalent cation concentrations on the chemical reactivity of the ps, aps, and ss species. Elevated NaCl concentration leads to a dramatic increase in the Os,py and KMnO4 modification of ss molecules and the ps, but not the aps, duplex. We attribute the apparent reaction with ps-DNA to a destabilization of this conformation under the conditions of reaction. In contrast, all reactions with DEPC are somewhat depressed at high salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reactivity of parallel-stranded DNA to chemical modification reagents. 227 21

A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized. Using the Chlamydomonas TAT with brain tubulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubulin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer.
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PMID:The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules. 373 80

The gene (AK) encoding adenylate kinase (AK) of Halobacterium halobium was cloned. AK consisted of 648 bp and coded for 216 amino acids (aa). S1 mapping and primer extension experiments indicated that the transcription start point (tsp) was located immediately upstream from the start codon. The TAT-like promoter sequence was found at a position 20-24 bp upstream from tsp. The most striking property of the enzyme was a putative Zn finger-like structure with four cysteines. It might contribute to the structural stability of the molecule in high-salt conditions. Phylogenetic analysis indicated two lineages of the AK family, the short and long types which diverged a long time ago, possibly before the separation of prokaryotes and eukaryotes. Although the H. halobium AK belongs to the long-type AK lineage, it is located in an intermediary position between the two lineages of the phylogenetic tree, indicating early divergence of the gene along the long-type lineage.
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PMID:Cloning and characterization of the gene encoding Halobacterium halobium adenylate kinase. 891 77

Neomycin is the most effective aminoglycoside (groove binder) in stabilizing a DNA triple helix. It stabilizes TAT, as well as mixed base DNA triplexes, better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (in the presence of salt), without any effect on the DNA duplex. (1) Triplex stabilization by neomycin is salt dependent (increased KCl and MgCl(2) concentrations decrease neomycin's effectiveness, at a fixed drug concentration). (2) Triplex stabilization by neomycin is pH dependent (increased pH decreases neomycin's effectiveness, at a fixed drug concentration). (3) CD binding studies indicate approximately 5-7 base triplets/drug apparent binding site, depending upon the structure/sequence of the triplex. (4) Neomycin shows nonintercalative groove binding to the DNA triplex, as evident from viscometric studies. (5) Neomycin shows a preference for stabilization of TAT triplets but can also accommodate CGC(+) triplets. (6) Isothermal titration calorimetry (ITC) studies reveal an association constant of approximately 2 x 10(5) M(-)(1) between neomycin and an intramolecular triplex and a higher K(a) for polydA.2polydT. (7) Binding/modeling studies show a marked preference for neomycin binding to the larger W-H groove. Ring I/II amino groups and ring IV amines are proposed to be involved in the recognition process. (8) The novel selectivity of neomycin is suggested to be a function of its charge and shape complementarity to the triplex W-H groove, making neomycin the first molecule that selectively recognizes a triplex groove over a duplex groove.
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PMID:Neomycin binding to Watson-Hoogsteen (W-H) DNA triplex groove: a model. 1265 3

DNA triplexes have been the subject of great interest due to their ability to interfere with gene expression. The inhibition of gene expression involves the design of stable triplexes under physiological conditions; therefore, it is important to have a clear understanding of the energetic contributions controlling their stability. We have used a combination of UV spectroscopy and differential scanning calorimetric (DSC) techniques to investigate the unfolding of intramolecular triplexes, d(A(n)C5T(n)C5T(n)), where n is 5-7, 9, and 11, and related triplexes with a single AT --> TA substitution in their duplex stem. Specifically, we obtain standard thermodynamic profiles for the unfolding of each triplex in buffer solutions containing 0.1 M or 1 M NaCl. The triplexes unfold in monophasic or biphasic transitions (triplex --> duplex --> coil) depending on the concentration of salt used and position of the substitution, and their transition temperatures are independent of strand concentration. The DSC curves of the unsubstituted triplexes yielded an unfolding heat of 13.9 kcal/mol for a TAT/TAT base-triplet stack and a heat capacity of 505 cal/ degrees C.mol. The incorporation of a single substitution destabilizes triplex formation (association of the third strand) to a larger extent in 0.1 M NaCl, and the magnitude of the effects also depends on the position of the substitution. The combined results show that a single AT --> TA substitution in a homopurine/homopyrimidine duplex does not allow triplex formation of the neighboring five TAT base triplets, indicating that the in vivo formation of triplexes, such as H-DNA, is exclusive to homopurine/homopyrimidine sequences.
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PMID:Calorimetric unfolding of intramolecular triplexes: length dependence and incorporation of single AT --> TA substitutions in the duplex domain. 1685 34

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.
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PMID:A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes. 1733 52

Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of deltaPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using deltaV1-1 - a selective peptide inhibitor of deltaPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or deltaV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the deltaV1-1-treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with deltaV1-1 was also associated with decreased deltaPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with deltaV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that deltaPKC may specifically act to dysregulate tight junction components. Together, these results suggest that deltaPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.
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PMID:Sustained pharmacological inhibition of deltaPKC protects against hypertensive encephalopathy through prevention of blood-brain barrier breakdown in rats. 1809 80

MYB-type transcription factors contain the conserved MYB DNA-binding domain of approximately 50 amino acids and are involved in the regulation of many aspects of plant growth, development, metabolism and stress responses. From soybean plants, we identified 156 GmMYB genes using our previously obtained 206 MYB unigenes, and 48 were found to have full-length open-reading frames. Expressions of all these identified genes were examined, and we found that expressions of 43 genes were changed upon treatment with ABA, salt, drought and/or cold stress. Three GmMYB genes, GmMYB76, GmMYB92 and GmMYB177, were chosen for further analysis. Using the yeast assay system, GmMYB76 and GmMYB92 were found to have transactivation activity and can form homodimers. GmMYB177 did not appear to have transactivation activity but can form heterodimers with GmMYB76. Yeast one-hybrid assay revealed that all the three GmMYBs could bind to cis-elements TAT AAC GGT TTT TT and CCG GAA AAA AGG AT, but with different affinity, and GmMYB92 could also bind to TCT CAC CTA CC. The transgenic Arabidopsis plants overexpressing GmMYB76 or GmMYB177 showed better performance than the GmMYB92-transgenic plants in salt and freezing tolerance. However, these transgenic plants exhibited reduced sensitivity to ABA treatment at germination stage in comparison with the wild-type plants. The three GmMYB genes differentially affected a subset of stress-responsive genes in addition to their regulation of a common subset of stress-responsive genes. These results indicate that the three GmMYB genes may play differential roles in stress tolerance, possibly through regulation of stress-responsive genes.
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PMID:Soybean GmMYB76, GmMYB92, and GmMYB177 genes confer stress tolerance in transgenic Arabidopsis plants. 1872 8

Heart failure (HF) is a chronic syndrome in which pathological cardiac remodeling is an integral part of the disease and mast cell (MC) degranulation-derived mediators have been suggested to play a role in its progression. Protein kinase C (PKC) signaling is a key event in the signal transduction pathway of MC degranulation. We recently found that inhibition of epsilonPKC slows down the progression of hypertension-induced HF in salt-sensitive Dahl rats fed a high-salt diet. We therefore determined whether epsilonPKC inhibition affects MC degranulation in this model. Six week-old male Dahl rats were fed with a high-salt diet to induce systemic hypertension, which resulted in concentric left ventricular hypertrophy at the age of 11 weeks, followed by myocardial dilatation and HF at the age of 17 weeks. We administered epsilonV1-2, an epsilonPKC-selective inhibitor peptide (3 mg/kg/day), deltaV1-1, a deltaPKC-selective inhibitor peptide (3 mg/kg/day), TAT (negative control; at equimolar concentration; 1.6 mg/kg/day) or olmesartan (angiotensin receptor blocker [ARB] as a positive control; 3 mg/kg/day) between 11 weeks and 17 weeks. Treatment with epsilonV1-2 attenuated cardiac MC degranulation without affecting MC density, myocardial fibrosis, microvessel patency, vascular thickening and cardiac inflammation in comparison to TAT- or deltaV1-1-treatment. Treatment with ARB also attenuated MC degranulation and cardiac remodeling, but to a lesser extent when compared to epsilonV1-2. Finally, epsilonV1-2 treatment inhibited MC degranulation in isolated peritoneal MCs. Together, our data suggest that epsilonPKC inhibition attenuates pathological remodeling in hypertension-induced HF, at least in part, by preventing cardiac MC degranulation.
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PMID:Mast cells and epsilonPKC: a role in cardiac remodeling in hypertension-induced heart failure. 1880 78

We report herein a simple, inexpensive fabrication methodology of salt microwells, and define the utility of the latter as nanoparticle containers for highly sensitive surface-enhanced Raman scattering (SERS) studies. AFM characterization of Ag and Au loaded salt microwells reveal the ability to contain favorable nanostructures such as nanoparticle dimers, which can significantly enhance the Raman intensity of molecules. By performing diffraction-limited confocal Raman microscopy on salt microwells, we show high sensitivity and fidelity in the detection of dyes, peptides, and proteins, as a proof of our concept. The SERS limit of detection (accumulation time of 1 s) for rhodamine B and TAT contained in salt mircowells is 10 pM and 1 nM, respectively. The Raman characterization measurements of salt microwells with three different laser lines (532 nm, 632.81 nm, 785 nm) reveal low background intensity and high signal-to-noise ratio upon nanoparticle loading, which makes them suitable for enhanced Raman detection. SERS mapping of these sub-femtoliter containers show spatial confinement of the relevant analyte to a few microns, which make them potential candidates for microscale bioreactors.
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PMID:SERS in salt wells. 1975 May 33


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