Theory of non-stationary activated rate processes : nonexponential relaxation kinetics

We have explored a simple microscopic model to simulate a thermally activated rate process where the associated bath which comprises a set of relaxing modes is not in an equilibrium state. The model captures some of the essential features of non-Markovian Langevin dynamics with a fluctuating barrier. Making use of the Fokker-Planck description we calculate the barrier dynamics in the steady state and non-stationary regimes. The Kramers-Grote-Hynes reactive frequency has been computed in closed form in the steady state to illustrate the strong dependence of the dynamic coupling of the system with the relaxing modes. The influence of nonequilibrium excitation of the bath modes and its relaxation on the kinetics of activation of the system mode is demonstrated. We derive the dressed time-dependent Kramers rate in the nonstationary regime in closed analytical form which exhibits strong non-exponential relaxation kinetics of the reaction co-ordinate. The feature can be identified as a typical non-Markovian dynamical effect.Comment: Plain Latex, no figure, 31 pages, to appear in J. Chem. Phy

Increased sensitivity of African American triple negative breast cancer cells to nitric oxide-induced mitochondria-mediated apoptosis.

BackgroundBreast cancer is a complex heterogeneous disease where many distinct subtypes are found. Younger African American (AA) women often present themselves with aggressive form of breast cancer with unique biology which is very difficult to treat. Better understanding the biology of AA breast tumors could lead to development of effective treatment strategies. Our previous studies indicate that AA but not Caucasian (CA) triple negative (TN) breast cancer cells were sensitive to nitrosative stress-induced cell death. In this study, we elucidate possible mechanisms that contribute to nitric oxide (NO)-induced apoptosis in AA TN breast cancer cells.MethodsBreast cancer cells were treated with various concentrations of long-acting NO donor, DETA-NONOate and cell viability was determined by trypan blue exclusion assay. Apoptosis was determined by TUNEL and caspase 3 activity as well as changes in mitochondrial membrane potential. Caspase 3 and Bax cleavage, levels of Cu/Zn superoxide dismutase (SOD) and Mn SOD was assessed by immunoblot analysis. Inhibition of Bax cleavage by Calpain inhibitor, and levels of reactive oxygen species (ROS) as well as SOD activity was measured in NO-induced apoptosis. In vitro and in vivo effect of NO treatment on mammary cancer stem cells (MCSCs) was assessed.Results and discussionNO induced mitocondria-mediated apoptosis in all AA but not in CA TN breast cancer cells. We found significant TUNEL-positive cells, cleavage of Bax and caspase-3 activation as well as depolarization mitochondrial membrane potential only in AA TN breast cancer cells exposed to NO. Inhibition of Bax cleavage and quenching of ROS partially inhibited NO-induced apoptosis in AA TN cells. Increase in ROS coincided with reduction in SOD activity in AA TN breast cancer cells. Furthermore, NO treatment of AA TN breast cancer cells dramatically reduced aldehyde dehydrogenase1 (ALDH1) expressing MCSCs and xenograft formation but not in breast cancer cells from CA origin.ConclusionsEthnic differences in breast tumors dictate a need for tailoring treatment options more suited to the unique biology of the disease

Stage-specific expressions of four different ribonuclease H genes in Leishmania

The human pathogen of the genus Leishmania cause worldwide morbidity and infection of visceral organs by some species may be lethal. Lack of rational chemotherapy against these pathogens dictates the study of differential biochemistry and molecular biology of the parasite as compared to its human host. The ubiquitous enzyme ribonuclease H (RNase H, EC 3.1.26.4) cleaves the RNA from a RNA:DNA duplex and is critical for the replication of DNA in the nucleus and the mitochondria. We have characterized four RNase H genes from Leishmania: one is of type I (LRNase HI) and three others are of type II (LRNase HIIA, -HIIB and -HIIC). In contrast human cells have only one type I and one type II RNase H. All the four RNase H genes in Leishmania are single copy and located in discrete chromosomes. When expressed inside RNase H-deficient E. coli, all of the four Leishmania RNase H were capable to complement the genetic defect of the E. coli, indicating their identity as RNase H. The enzymes are differentially expressed in the promastigotes and the amastigotes, the forms that thrives in entirely different physico-chemical conditions in nature. Nucleotide sequences of the 5'-UTRs of three of these mRNAs have upstream open reading frames. Understanding the regulation of these four distinct ribonuclease H genes in Leishmania will help us better understand leishmanial parasitism and may help us to design rational chemotherapy against the pathogen

Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>BRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study.</p> <p>Results</p> <p>Human <it>BRCA2 </it>gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G₂), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm.</p> <p>Conclusions</p> <p><it>BRCA2 </it>gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter <it>in vivo </it>and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.</p

Quantum theory of dissipation of a harmonic oscillator coupled to a nonequilibrium bath; Wigner-Weisskopf decay and physical spectra

We extend the quantum theory of dissipation in the context of system-reservoir model, where the reservoir in question is kept in a nonequilibrium condition. Based on a systematic separation of time scales involved in the dynamics, appropriate generalizations of the fluctuation-dissipation and Einstein's relations have been pointed out. We show that the Wigner-Weisskopf decay of the system mode results in a rate constant which depending on the relaxation of nonequilibrium bath is dynamically modified. We also calculate the time-dependent spectra of a cavity mode with a suitable gain when the cavity is kept in contact with a nonequilibrium bath.Comment: Plain Latex, 28 pages, 2 PS figure

Alternative initiation and splicing in dicer gene expression in human breast cells

INTRODUCTION: Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene. METHOD: We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression in vitro and in vivo. RESULTS: We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein. CONCLUSION: Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells