Theoretical study of molecular recognition by Hoechst 33258 derivatives

The factors responsible for the binding of Hoechst 33258 with DNA residues have been investigated in this work using the AM1 method. First and foremost, it is found that, although all crystal structure determinations indicate a preference for binding at AT rich sites, the hydrogen bond strength is actually greater for complexes with cytosine and guanine. From this, it has been inferred that other factors such as electrostatic, van der Waals interactions and nonbonded contacts with the walls of the minor groove have a strong role to play in the binding process. The hydrogen bond is found to be stronger for complexation with the thymine O2 than with the adenine N3, in line with experimental observations. Combined QM/MM studies on the drug complexed with the Dickerson-Drew dodecamer reveal that binding induces structural changes in both the ligand as well as DNA. Electron donating substituents at the para position in the phenyl ring of Hoechst 33258 lead to stronger binding with DNA. A correlation with the octanol/water partition coefficients points to the importance of hydrophobic and electrostatic interactions.     

Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fibrosis bronchial epithelial cells

4-Phenylbutyrate (4-PBA) is an oral butyrate derivative that has recently been approved for treatment of urea cycle disorders and is under investigation in clinical trials of cancer, hemoglobinopathies, and cystic fibrosis (CF). We hypothesized that proteome profiling of IB3-1 cystic fibrosis bronchial epithelial cells treated with 4-PBA would identify butyrate-responsive cellular chaperones, protein processing enzymes, and cell trafficking molecules associated with the amelioration of the chloride transport defect in these cells. Protein profiles were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Over a pI range of 4-7 and molecular weight from 20 to 150 kDa a total of 85 differentially expressed proteins were detected. Most of the identified proteins were chaperones, catalytic enzymes, and proteins comprising structural elements, cellular defense, protein biosynthesis, trafficking activity, and ion transport. Subsets of these proteins were confirmed by immunoblot analysis. These data represent a first-draft of the pharmacoproteomics map of 4-PBA treated cystic fibrosis bronchial epithelial cells.     

Pro-urokinase-type plasminogen activator is a substrate for hepsin

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1') peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 x 10(5) m(-1) s(-1)) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 +/- 1.9 nm uPA h(-1), which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.     

Integration Host Factor of Mycobacterium tuberculosis,mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.     

Protein metabolism during natural ageing in desiccating recalcitrant seeds of Shorea robusta

Seeds of Shorea robusta (sal) are recalcitrant owing to its high desiccation sensitivity. Germinability in sal seed was lost rapidly from 100 to 0 % within 8 days. Protein oxidation examined separately in axis and cotyledon of ageing sal seeds by monitoring the levels of carbonyls, hydroperoxide, malondialdehyde and 4-hydroxy-2-nonenal adducts with protein, Amadori and Maillard reaction products. Changes in protease and proteasome activity were also estimated. The levels of all the modified proteins and activities of protease and proteasome were similar in axis and cotyledons. The amounts of carbonyls (5.5 fold in axis and 3.9 fold in cotyledons) and hydroperoxides (13.5 fold in axis and 12 fold in cotyledons) increased significantly as the seeds became non-viable. Similarly, the levels of malondialdehyde and 4-hydroxy-2-nonenal adducts promoted as the storage period advanced and reached tenfold both in the axis and cotyledons in non-viable seeds. The ageing also promoted levels of reducing sugar along with rapid enhancement in the levels of Amadori and Maillard reaction products, respectively, by 4.4 and 1.8 fold in 5 days sal seeds. Substantial promotion in protease activity both in the axis (sevenfold) and cotyledons (tenfold) of absolutely aged seeds was discernible. The activity of proteasome exhibited steady decline from 0.767 to 0.170 nmol min^?1 g^?1 DM in axis and 0.20–0.086 nmol min^?1 g^?1 DM in cotyledons of ageing sal seeds. Changes in the ROS and protein catabolism/oxidation have been discussed to establish loss of germinability in desiccating recalcitrant sal seeds.     

Molecular Characterization of Primary Gene Pool of Chickpea Based on ISSR Markers

Genetic diversity and relationships within and among members of the primary gene pool of chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum,, and four of C. echinospermum, were investigated using 31 ISSR markers. The study revealed moderate diversity, detecting 141 fragments, of which 79 (56%) were polymorphic. Averages were 0.125 for polymorphic information content, 0.350 for marker index, and 0.715 for resolving power. The UPGMA dendrogram and the principal coordinate analysis revealed a clear differentiation between wild and cultivated accessions. The clustering pattern did not strictly follow the grouping of accessions by geographic origin but was in good agreement with the pedigree data and the seed type. The study demonstrates that ISSRs provide promising marker tools in revealing genetic diversity and relationships in chickpea and can contribute to efficient identification, conservation, and utilization of germplasm for plant improvement through conventional as well as molecular breeding approaches.