Reductive inactivation of digitoxin by Eubacterium lentum cultures

The obligate anaerobe Eubacterium lentum inactivated the cardiac glycoside digitoxin by reducing the double bond in the lactone ring. This conversion was quantitative when the substrate was incubated at a concentration of 10 micrograms/ml. The reduction reaction coincided with the growth phase of the bacterium. The stereochemical configuration at C-20 of the reduction product dihydrodigitoxin was found to be R. Incubation of digitoxigenin and its mono- and bisdigitoxosides individually with E. lentum led to the formation of their respective dihydro derivatives. The configuration at C-20 of these reduced metabolites was also found to be R.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Wrinkled DNA.

The B form of poly d(GC):poly d(GC) in orthorhombic microcrystallites in oriented fibers has a secondary structure in which a dinucleotide is the repeated motif rather than a mononucleotide as in standard, smooth B DNA. One set of nucleotides (probably GpC) has the same conformations as the smooth form but the alternate (CpG) nucleotides have a different conformation at C3'-O3'. This leads to a distinctive change in the orientation of the phosphate groups. Similar perturbations can be detected in other poly d(PuPy):poly d(PuPy) DNAs such as poly d(IC):poly d(IC) and poly d(AT):poly d(AT) in their D forms which have tetragonal crystal environments. This suggests that such perturbations are intrinsic to all stretches of duplex DNA where purines and pyrimidines alternate and may play a role in the detection and exploitation of such sequences by regulatory proteins.     

Conformation of cyclic dipeptides from empirical energy calculations

Empirical energy calculations on cyclo-Gly-X with X- Phe, Tyr, Val, and Leu as a function of the side-chain torsion angles χ indicate that the conformation of minimum energy are characterized by χ₁ = 60°, χ² = 90° for Phe and Try, χ¹ = ?60° for Val and χ¹ = ?60°, χ² = 180° and χ¹ = 60° and χ² = 150° for Leu. The minimum energy conformation of cyclo-Gly-Phe and cyclo-Gly-Val have the side chains of Phe and Val stacked over the poperazinedione ring as suggested by NMR and found for cyclo-Gly-Tyr crystal structure. In contrast, the Leu side chain is expected to exist in an extended or a quasi-folded form.     

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal ß-Gal residueswas investigated by screening sulfotransferase activity presentin 37 human tissue specimens toward the following synthesizedacceptor moieties: Galß1,3GalNAc-O-Al, Galß1,4GlcNAcß-O-Al,Galß1,3GlcNAcß-O-Al, and mucin-type Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnstructures containing a C-3 methyl substituent on either Gal.Two distinct types of Gal: 3-O-sulfotransferases were revealed.One (Group A) was specific for the Galß1, 3GalNAc-linkage and the other (Group B) was directed toward the Galß1,4GlcNAcbranch ß1,6 linked to the blood group T hapten. Enzymeactivities found in breast tissues were unique in showing astrict specificity for the T-hapten. Galß-O-allylor benzyl did not serve as acceptors for Group A but were veryactive with Group B. An exainination of activity present insix human sera revealed a specificity of the serum enzyme towardß1,3 linked Gal, particularly, the T-hapten withoutß1,6 branching. Group A was highly active toward T-haptenlacrylamidecopolymer, anti-freeze glycoprotein, and fetuin O-glycosidicasialo glycopeptide; less active toward fetuin triantennaryasialo glycopeptide; and least active toward bovine IgG diantennaryglycopeptide. Group B was moderately and highly active, respectively,with the latter two glycopeptides noted and least active withthe first two. Competition experiments performed with Galß1,3GaLNAc-O-Aland Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnhaving a C-3 substituent (methyl or sulfate) on either Gal reinforcedearlier findings on the specificity characteristics of GroupA and Group B. Group A displayed a wider range of optimal activity(pH 6.0–7.4), whereas Group B possessed a peak of activityat pH 7.2. Mg²⁺ stimulated Group A 55% and Group B 150%, whereasMn⁺² stimulated Group B 130% but inhibited Group A 75%. Ca²⁺stimulated Group B 100% but inhibited Group A 35%. Group A andGroup B enzymes appeared to be of the same molecular size (<100,000Da) as observed by Sephacryl S-100 HR column chromatography.The following effects upon Gal: 3-O- sulfotransferase activitiesby fucose, sulfate, and other substituents on the carbohydratechains were noted. (1) A methyl or GlcNAc substituent on C-6of GalNAc diminished the ability of Galß1,3GalNAc-O-Alto act as an acceptor for Group A. (2) An 1,3-fucosyl residueon the ß1,6 branch in the mucin core structure didnot affect the activity of Group A toward Gal linked ß1,3to GalNAc-. (3) Lewis x and Lewis a terminals did not serveas acceptors for either Group A or B enzymes. (4) Eliminationof Group B activity on Gal in the ß1,6 branch owingto the presence of a 3-fucosyl or 6-sulfo group on GlcNAc didnot hinder any action toward Gal linked ß1,3 to GalNAc.(5) Group A activity on Gal linked ß1,3 to GalNAcremained imaffected by 3'-sulfation of the ß1,6 branch.The reverse was true for Group B. (6) The acceptor activityof the T-hapten was increased somewhat upon C-6 sulfation ofGalNAc, whereas, C-6 slalylation resulted in an 85% loss ofactivity. (7) A novel finding was that Galß1,4GlcNAcß-O-Aland Galß1,3GlcNAcß-O-M, upon C-6 sulfationof the GlcNAc moiety, became 100% inactive and 5- to 7-foldactive, respectively, in their ability to serve as acceptorsfor Group B. human tissues glycoprotein galactose:sulfotransferase specificities kinetic properties     

Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells.

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.     

Discovery of potent inhibitors for interleukin-2-inducible T-cell kinase: structure-based virtual screening and molecular dynamics simulation approaches

In our study, a structure-based virtual screening study was conducted to identify potent ITK inhibitors, as ITK is considered to play an important role in the treatment of inflammatory diseases. We developed a structure-based pharmacophore model using the crystal structure (PDB ID: 3MJ2) of ITK complexed with BMS-50944. The most predictive model, SB-Hypo1, consisted of six features: three hydrogen-bond acceptors (HBA), one hydrogen-bond donor (HBD), one ring aromatic (RA), and one hydrophobic (HY). The statistical significance of SB-Hypo1 was validated using wide range of test set molecules and a decoy set. The resulting well-validated model could then be confidently used as a 3D query to screen for drug-like molecules in a database, in order to retrieve new chemical scaffolds that may be potent ITK inhibitors. The hits retrieved from this search were filtered based on the maximum fit value, drug-likeness, and ADMET properties, and the hits that were retained were used in a molecular docking study to find the binding mode and molecular interactions with crucial residues at the active site of the protein. These hits were then fed into a molecular dynamics simulation to study the flexibility of the activation loop of ITK upon ligand binding. This combination of methodologies is a valuable tool for identifying structurally diverse molecules with desired biological activities, and for designing new classes of selective ITK inhibitors.

Endothelial nitric oxide synthase gene polymorphisms and the risk of acute myocardial infarction in a South Indian population

Myocardial infarction (MI) is a complex multi-factorial, polygenic disorder which results from an interaction between a person’s genetic makeup and various environmental factors. Nitric oxide (NO), a potent vasodilator produced by endothelial cells, plays an important role in the regulation of blood pressure, regional blood flow and also inhibits platelet aggregation, vascular smooth muscle cell proliferation and leukocyte adhesion to vascular endothelium. Our aim was to analyze the association of NOS3 (endothelial nitric oxide synthase 3) 894G>T and ?786T>C gene polymorphisms and MI risk in the South Indian population. A total of 287 MI patients, 279 risk control patients and 321 healthy controls were recruited for the retrospective study. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There was no significant association observed between NOS3 894G>T, ?786T>C polymorphisms and MI. A significant difference was observed in the distribution of GT genotype of the NOS3 894G>T polymorphism between the cases and the risk controls (p = 0.05) but the odds ratio (0.6) did not show risk for MI. The present study showed lack of association between NOS3 gene polymorphisms and MI in South Indian population.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD⁺-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD⁺ and acetylated lysine) and inhibitor (sirtinol) in the NAD⁺ binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5?ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.