A ~35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP

BACKGROUND: The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested. RESULTS: Partially purified fractions containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. However, further purification revealed the presence of a putative 35 kDa insect cell protein (p35) which was found responsible for the DNA binding activity. A close match to the 9/11 bases of the ARS consensus sequence was sufficient for p35 binding activity. A DNA fragment from the human c-myc origin region containing yeast ACS like elements also showed p35 binding activity. CONCLUSIONS: We have identified a Spodoptera frugiperda protein with ATP dependent DNA binding activity to ACS like elements. ACS like elements have been reported to be essential for ORC binding and replication initiation in yeast but their role in higher eukaryotes still remains elusive. Like the ARS consensus sequence elements of yeast, ACS like elements found in c-myc and lamin beta 2 origin regions may play similar roles in replication and indicate a conserved role for this DNA motif among eukaryotes

Current Management Strategies in Breast Cancer by Targeting Key Altered Molecular Players

Breast Cancer is second largest disease affecting women worldwide. It remains the most frequently reported and leading cause of death among women in both developed and developing countries. Chemoprevention is one the promising approaches which reduces breast cancer. Tamoxifen and raloxifene are commonly used for treatment of breast cancer in women with high risk, although resistance occurs by tamoxifen after five years of therapy and both drugs cause uterine cancer and thromboembolic events. Aromatase inhibitors are coming up as potential option for prevention in treatment with adjuvant trials in practice. The combination of aromatase inhibitors along with tamoxifen can also be beneficial. For this, clinical trials based on large number of patients with optimal dose and lesser side effects have to be more in practice. Despite the clinical trials going on, there is need of better molecular models which can identify high risk population and new agents with better benefit having less side effects and improved biomarkers for treating breast cancer

Graphene Oxide-Silver Nanocomposite Induced Apoptosis in Human Hepatoma (HepG2) Cells Through Oxidative Stress and Caspase Dependent Signalling Pathway

298-304At present, the nanotechnology-based therapeutic agents have been emerged as the most reliable tool for various dangerous disorders including cancer. Liver cancer is one of the top five deadliest cancers. Hepatocellular Carcinoma (HCC) is the third most leading cause of death after lung and stomach cancer. Graphene Oxide, due to its unique chemical, mechanical, and optical properties, has offered a wide range of applications in biomedical fields. Silver nanoparticles can easily enter into mammalian cells, accumulate in the macrophages, and interact with biological molecules. In the present investigation, the effects of Graphene Oxide-Ag nanocomposites on human Hepatoma Cells (HepG2) have been investigated. Graphene oxide has been synthesised by modified Hummer’s method and further, Graphene Oxide-Ag nanocomposites have been prepared. The effect of nanoparticles on cell viability has been observed. For understanding the molecular pathway, the effects on oxidative stress (ROS and GSH levels), Caspase-3 Activity and Apoptotic Cell Population have been examined. Further, to confirm the role of oxidative stress and caspase-3 activity, the cell viability has been measured in the presence or absence of specific inhibitors. The results demonstrate that GO-Ag nanocomposites induce cytotoxicity, oxidative stress and apoptosis in HepG2 cells through caspase dependent pathway. The oxidative stress plays a crucial role in GO-Ag nanocomposites induced caspase dependent apoptosis. Thus, it can be concluded that GO-Ag nanocomposites show therapeutic efficacy in cancer cells

A Unique 45-Amino-Acid Region in the Toprim Domain of Plasmodium falciparum Gyrase B Is Essential for Its Activity▿ †

DNA gyrase is the only topoisomerase that can introduce negative supercoils into the DNA at the cost of ATP hydrolysis. Some but not all the steps of the topoisomerization reaction are understood clearly for both eukaryotic topoII and DNA gyrase. This study is an attempt to understand whether the B subunit of DNA gyrase binds to DNA directly, which may be central to the stimulation of its ATPase activity essential for gyrase function. We have dissected the Plasmodium falciparum gyrase B (PfGyrB) subunit to identify a 45-amino-acid region in the toprim domain that is responsible for its intrinsic DNA binding activity, DNA-stimulated ATPase activity, and DNA cleavage. We find that DNA has to enter through the ATP-operated clamp of PfGyrB to gain access to the DNA binding region. Furthermore, the rate of ATP hydrolysis of PfGyrB increases significantly with increasing DNA length, suggesting a possible communication between the ATPase domain and the DNA binding region that can account for its optimal ATPase activity. These results not only highlight the mechanism of GyrB action in the deadly human parasite P. falciparum but also provide meaningful insights into the current mechanistic model of DNA transport by gyrase during the topoisomerization reaction

Molecular Cloning of Apicoplast-Targeted Plasmodium falciparum DNA Gyrase Genes: Unique Intrinsic ATPase Activity and ATP-Independent Dimerization of PfGyrB Subunit

DNA gyrase, a typical type II topoisomerase that can introduce negative supercoils in DNA, is essential for replication and transcription in prokaryotes. The apicomplexan parasite Plasmodium falciparum contains the genes for both gyrase A and gyrase B in its genome. Due to the large sizes of both proteins and the unusual codon usage of the highly AT-rich P. falciparum gyrA (PfgyrA) and PfgyrB genes, it has so far been impossible to characterize these proteins, which could be excellent drug targets. Here, we report the cloning, expression, and functional characterization of full-length PfGyrB and functional domains of PfGyrA. Unlike Escherichia coli GyrB, PfGyrB shows strong intrinsic ATPase activity and follows a linear pattern of ATP hydrolysis characteristic of dimer formation in the absence of ATP analogues. These unique features have not been reported for any known gyrase so far. The PfgyrB gene complemented the E. coli gyrase temperature-sensitive strain, and, together with the N-terminal domain of PfGyrA, it showed typical DNA cleavage activity. Furthermore, PfGyrA contains a unique leucine heptad repeat that might be responsible for dimerization. These results confirm the presence of DNA gyrase in eukaryotes and confer great potential for drug development and organelle DNA replication in the deadliest human malarial parasite, P. falciparum

Design, synthesis and biological evaluation of quinazolin-4(3H)-one Schiff base conjugates as potential antiamoebic agents

In an effort to develop novel antiamoebic scaffolds having better efficacy than the standard drug metronidazole (IC50 = 1.80 μM) used against Entamoeba histolytica, quinazolin-4(3H)-one Schiff base conjugates were synthesized and evaluated against HM1: IMSS strain of E. histolytica. Out of the thirteen compounds (S2-S14), six compounds (S2, S3, S4, S5, S6 and S11) were found to be better inhibitors than metronidazole and showed low cytotoxicity on HeLa cells, a cervical cancer cell line. The structure of intermediate compound S1 was confirmed by crystal structure studies

Elongation by RNA polymerase II on chromatin templates requires topoisomerase activity

Transcription on chromatin by RNA polymerase II (pol II) is repressed as compared with transcription on histone-free DNA. In this study, we show that human topoisomerase I (topo I) and yeast topoisomerase II (topo II), each of which relax both positive and negative superhelical tension, reverse the transcriptional repression by chromatin. In the presence of bacterial topo I, which can relax only negative superhelical tension, the transcription is repressed on chromatin templates. The data together show that the relaxation of positive superhelical tension by these enzymes was the key property required for RNA synthesis from chromatin templates. In the absence of topoisomerase, transcriptional repression on chromatin depended on RNA length. The synthesis of transcripts of 100 nt or shorter was unaffected by chromatin, but repression was apparent when the RNA transcript was 200 nt or longer. These findings suggest that transcription on chromatin templates results in the accumulation of positive superhelical tension by the elongating polymerase, which in turn inhibits further elongation in the absence of topoisomerase activity