GST-Induced dimerization of DNA-binding domains alters characteristics of their interaction with DNA

The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the induction and modulation of morphogenetic events throughout Drosophila melanogaster development. Two members of the nuclear receptor superfamily, the product of the EcR (EcR) and of the ultraspiracle genes (Usp), heterodimerize to form its functional receptor. To study the receptor-DNA interaction, critical for regulating 20E-dependent gene expression, it is necessary to produce large quantities of EcR and Usp DNA-binding domains. Toward this end DNA-binding domains of EcR and Usp (EcRDBD and UspDBD, respectively) were cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). However, the results of DNA-binding studies obtained with purified GST-DBDs were found to be questionable because the fused proteins oligomerized in solution due to the presence of GST. Therefore DBDs were released from GST-chimeric proteins by thrombin cleavage and then purified by glutathione-Sepharose 4B chromatography and by gel filtration on Superdex 75 HR. The gel mobility-shift experiments showed that UspDBD exhibited higher affinity than EcRDBD toward a 20-hydroxyecdysone response element from the Drosophila hsp 27 gene (hsp 27pal). Furthermore, formation of the heterodimeric EcRDBD-UspDBD complex was observed to be synergistic when equimolar mixture of both DBDs was incubated with hsp 27pal. Surprisingly, GST-EcRDBD bound hsp 27pal with higher affinity than GST-UspDBD. This difference was accompanied by the impaired ability of the GST-DBDs to interact synergistically with hsp 27pal. This is the first report on expression and purification of the soluble DBDs of the functional ecdysteroid receptor with satisfying yields. Furthermore, our results add to the recent findings which indicate the need for caution in interpreting the activities of GST fusion proteins.     

Calmodulin is essential for estrogen receptor interaction with its motif and activation of responsive promoter

Calmodulin (CaM) has been reported to have affinity for the estrogen receptor (ER). Observations reported here reveal a direct physical interaction between purified CaM and ER. This direct ER-CaM interaction may be an initial event preceding the assembly of ER plus auxiliary proteins into the active ER complex with its DNA motif, the estrogen response element. We demonstrate that CaM is an integral component of this complex by using a system reconstituted from purified ER and nuclear extract from ER-negative breast cancer cells and also with ER-depleted nuclear extract of an ER-positive breast cancer cell line. Although CaM is essential for formation of this complex, it is not sufficient, suggesting roles also of auxiliary proteins. CaM also is functionally required for activation of an ER-responsive promoter, in the 17beta-estradiol-ER pathway of hormone action and regulation of 17beta-estradiol-responsive gene expression that is associated with proliferation of mammary epithelial cells.     

An orphan nuclear receptor lacking a zinc-finger DNA-binding domain: interaction with several nuclear receptors

BACKGROUND: Affections of the lacrimal drainage system are often seen by ophthalmologists. Inspection, palpation, diagnostic rinsing and sounding can distinguish anatomical stops before or after tear sac. For final diagnostics however more apparative examinations are necessary. The ultrasonic examination with contrast media is a simple method for diagnostics of affections of the lacrimal drainage system besides the dacryocystography, the scintigraphy and other ones. PATIENTS AND METHODS: On 12 patients with a dysfunctional lacrimal drainage system and 12 normal controls the ultrasonic examination with instillation of contrast media: silicon oil, glycerine, dispersions of almond oil and viscoelastic substances was performed. All examinations were performed with the 13 B-scan. RESULTS: The lacrimal drainage way was detectable from the canaliculus to the middle nasolacrimal duc CONCLUSION: An additional advantage of the ultrasonic examination with contrast-media is the neglect of radiation, the simple and often repeatable examination method, especially the enhancement of the contrast of different valves and stenoses and mucinous deposits in stationary anatomical variations and dynamic defects.     

An operational model of the antigen-antibody interaction

We investigated smooth muscle cell proliferation associated with restenosis after percutaneous transluminal coronary angioplasty (PTCA) in 8 arteries with fragmented internal elastic lamina obtained at autopsy in 7 patients who died between 2 months to 2 years 11 months after coronary angioplasty. The internal elastic lamina fragmentation, measured longitudinally along the blood vessels, measured 6.6 +/- 6.9 mm. Smooth muscle cell proliferation was concentrated around the fragmented internal elastic lamina, extending longitudinally even to unfragmented areas. The proliferation of smooth muscle cells extended for 1.8 +/- 2.2 mm in the proximal portion of the fragmentation, and for 2.0 +/- 2.9 mm in the distal portion. The possibility of new stenoses resulting from smooth muscle cell proliferation at sites adjacent to those subjected to PTCA should be borne in mind when PTCA of the proximal segments of the left anterior descending coronary artery is contemplated.     

The conformation of peptide thymosin alpha 1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. A possible model for its interaction with the lymphocyte membrane

The 28-residue peptide thymosin alpha1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin alpha1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin alpha1 has a structure characterized by two regions. A beta-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an alpha helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin alpha1.     

A conserved sequence motif in the integrin beta3 cytoplasmic domain is required for its specific interaction with beta3-endonexin

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.     

An experimental model for the rational treatment of arrhythmias: a clinical study with quinidine

Fourteen patients with supraventricular and/or ventricular ectopic beats selected by clinical, dynamic ECG and exercise test, underwent a basal continuous ECG recording (Holter) and then were given 400 mg of quinidine orally. The concentrations of the drug were determined in blood samples taken 30 minutes before, and 1, 2, 4, 6, 8, 12, and 24 hours after administration. On the same day patients were also monitored by continuous ECG recording (Holter). One week later, after a washout period, the same subjects were treated with 200 mg of quinidine orally 4 times a day for two weeks. On day 1, 2, 4, 7 and 14 quinidine was determined in plasma, and on day 7 and 14 a 24 hour ECG recording was also done. On the basis of pharmacokinetic parameters obtained from the acute test, the chronic levels of the drug were predicted. Predicted values were superimposable to those observed. Thus, the kinetics of single-dose quinidine is able to predict the steady-state levels after repeated dosing. During acute administration there was an increase in QTc interval and a decrease in ectopic beats. These effects correlated with a sigmoid pattern with acute quinidine levels. Concentrations producing 50% of the effect (EC50) could be calculated from these curves. In spite of similar steady-state blood levels of quinidine, some patients after chronic therapy did not respond to treatment (non responders). Non responders could be predicted by the acute test because they had greater EC50 values of QTc increase: patients with EC50 greater than 2 mg/L were all non responders.     

ParaMEME: a parallel implementation and a web interface for a DNA and protein motif discovery tool

Many advanced software tools fail to reach a wide audience because they require specialized hardware, installation expertise, or an abundance of CPU cycles. The worldwide web offers a new opportunity for distributing such systems. One such program, MEME, discovers repeated patterns, called motifs, in sets of DNA or protein sequences. This tool is now available to biologists over the worldwide web, using an asynchronous, single-program multiple-data version of the program called ParaMEME that runs on an Intel Paragon XP/S parallel computer at the San Diego Super-computer Center. ParaMEME scales gracefully to 64 nodes on the Paragon with efficiencies > 72% for large data sets. The worldwide web interface to ParaMEME accepts a set of sequences interactively from a user, submits the sequences to the Paragon for analysis, and e-mails the results back to the user. ParaMEME is available for free public use at http://@www.sdsc.edu/CompSci/Biomed/ MEME.     

Preparation of stereoregulated antisense oligodeoxyribonucleoside phosphorothioate and interaction with its complementary DNA and RNA

Diastereoisomeric specificity of oligodeoxyribonucleoside phosphorothioate (OPT) in DNA/OPT and RNA/OPT hybrid formation was investigated. The difference in the configuration between RRRR and SSSS was reflected in the conformation and the stability of the DNA/OPT and RNA/OPT hybrids. Therefore, findings of this report rationalize the antisense effect by non-stereoregulated OPT and the difference of diastereoisomerism in susceptibility to RNase H.     

An experimental study on interaction of nodular cast iron with CRT glass

In the manufacturing of glass components of cathode ray tubes, ductile cast iron rings are commonly used as an integral part of the mold assembly. The thermo-chemical interaction of molten glass with cast iron therefore becomes critical as far as the mold life and productivity is concerned. In this perspective, the present study is carried out to understand and analyze the chemical interaction of cast iron when immersed in molten glass. The experiments were conducted in the temperature range of 700°C to 900°C for selected time duration of 0 to 100 hours. The important observations include the formation of diffusion zone at the glass metal interface. SEM-EDS analysis revealed that diffusion of silicon and lead from glass takes place in diffusion zone with varying degree, depending on temperature and time. The hardness variation across the interface correlates well with the measured compositional changes. The plausible reasons for the measured hardness increase in the diffusion zone are also discussed.     

A model for the interaction of trifluoroethanol with peptides and proteins

Scaphoid or longitudinal arch pads are frequently prescribed pedorthics for foot and ankle rehabilitation. These pedorthics are reported to be effective in mechanically supporting the medial longitudinal arch while reducing plantar and medial soft tissue strain. The objective of this study was to measure alterations in ambulatory plantar pressure metrics in a group of adults secondary to scaphoid pad application. The biomechanical rationale of this study was that the geometry of foot contact would be altered secondary to foot inversion. Ten adult male subjects with biomechanically normal feet were evaluated during multiple trials. A Holter type microprocessor-based portable in-shoe plantar pressure data acquisition system was used to record the dynamic data. Pressures were recorded from eight discrete plantar locations at the hindfoot, midfoot, and forefoot regions of the insole. Statistically significant (p < or = 0.05) increases in peak pressures were seen laterally with scaphoid pad application, while significant decreases in peak pressures with pad usage occurred at the hallux and the calcaneal region of the foot. At the medial longitudinal arch, peak pressures increased from near 0 to 115.3 kPa, contact durations increased from near 0 to 438 ms, and pressure-time integrals increased from near 0 to 33.4 kPa.s.