Potential of cadmium sulphide nanorods as an optical microscopic probe to the folding state of cytochrome C

The folding behavior of cytochrome C (Cyt-C) conjugated with CdS nanorods (CdSnr) is amenable to monitoring by bright field microscopy, the porosity and percolating behavior of such protein conjugated nanoclusters depending on the folding history prior to the conjugation. The method has been used to predict the thermal melting behavior as well as guanidine hydrochloride induced unfolding of Cyt-C. Dynamic light scattering studies indicate that the size distribution of the nanoforms widens in presence of the protein. Furthermore, there is emergence of clusters with higher conductivity and altered zeta potential. Increase of second virial coefficient of CdS nanoforms in the presence of Cyt-C (obtained from static light scattering experiments) implies presence of protein coat over the hydrophobic nanosurface. The results are supported by morphological changes observed through scanning electron microscopy (SEM). Accordingly, the X-ray diffraction pattern shows a change of crystallographic orientations of CdSnr in presence of Cyt-C.     

Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.     

CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.     

Differential acetylcholinesterase activity in rat cerebrum, cerebellum and hypothalamus

Acetylcholinesterase (AChE) has been purified from three different regions of rat brain using Sephadex G 200 column. SDS PAGE (6%) showed single band for the purified AChE fractions. Purified and lyophilized AChE from different (NH4)2SO4 precipitated fractions of three brain parts were utilized for in vitro enzyme kinetics using Dimethoate (Dmt) as inhibitor. K(m) values for cerebellum and hypothalamus were almost similar whereas cerebrum showed a different K(m) value compared to other two regions. With the drug Rivastigmine it was found that % G1 and G4 forms of AChE in three different parts of brain are different.     

A novel mechanism of FSH regulation of DNA synthesis in the granulosa cells of hamster preantral follicles: involvement of a protein kinase C-mediated MAP kinase 3/1 self-activation loop

The objective was to reveal whether a protein kinase C (PKC [all isozymes])-mediated self-sustaining MAPK3/1 (3/1 extracellular signal regulated kinase 2/1, also known as ERK2/1) activation loop was necessary for FSH- or epidermal growth factor (EGF)-induced DNA synthesis in the granulosa cells of intact preantral follicles. For this purpose, hamster preantral follicles were cultured with FSH or EGF in the presence of selective kinase inhibitors FSH or EGF phosphorylated RAF1, MAP2K1, and MAPK3/1. However, a relatively higher dose of EGF was necessary to sustain the MAPK3/1 activity, which was essential for cyclin-dependent kinase 4 (CDK4) activation and DNA synthesis. In intact preantral follicles, FSH or EGF stimulated DNA synthesis only in the granulosa cells. Sustained activation of MAPK3/1 beyond 3 h was independent of EGFR kinase activity but dependent on PKC activity, which appeared to form a self-sustaining MAPK3/1 activation loop by activating RAF1, MAP2K1, and PLA2G4 (phospholipase A2 [all cytosolic isozymes]). Inhibition of PKC activity as late as 4 h after the administration of FSH or EGF arrested DNA synthesis, which corresponded with attenuated phosphorylation of RAF1 and MAPK3/1, thus suggesting an essential role of PKC in MAPK3/1 activation. Collectively, these data present a novel self-sustaining mechanism comprised of MAPK3/1, PLA2G4, PKC, and RAF1 for CDK4 activation leading to DNA synthesis in granulosa cells. Either FSH or EGF can activate the loop to activate CDK4 and initiate DNA synthesis; however, consistent with our previous findings, FSH effect seems to be mediated by EGF, which initiates the event by stimulating EGFR kinase.     

Infrared surface plasmon resonance: a novel tool for real time sensing of variations in living cells

We developed a novel surface plasmon resonance (SPR) method, based on Fourier transform infrared (FTIR) spectroscopy, as a label-free technique for studying dynamic processes occurring within living cells in real time. With this method, the long (micrometer) infrared wavelength produced by the FTIR generates an evanescent wave that penetrates deep into the sample. In this way, it enables increased depth of sensing changes, covering significant portions of the cell-height volumes. HeLa cells cultivated on a gold-coated prism were subjected to acute cholesterol enrichment or depletion using cyclodextrins. Cholesterol insertion into the cell plasma membrane resulted in an exponential shift of the SPR signal toward longer wavelengths over time, whereas cholesterol depletion caused a shift in the opposite direction. Upon application of the inactive analog alpha-cyclodextrin (alpha-CD), the effects were minimal. A similar trend in the SPR signal shifts was observed on a model membrane system. Our data suggest that FTIR-SPR can be implemented as a sensitive technique for monitoring in real time dynamic changes taking place in living cells.