Chemistry
Understanding the interactions among polyelectrolytes by using inorganic polyoxometalate molecular clusters as model systems
Nanometer-scaled polyoxometalate (POM) molecular clusters exist as hydrophilic, highly soluble macroions in water and other polar solvents. Very interestingly, they do not stay as discrete ions even in very dilute solutions. Instead, we find that they universally tend to self-assemble into highly stable, monodispersed, hollow, spherical, single-layered shell-like structures (we call them “blackberries”), by using laser light scattering, TEM, SEM and SAXS studies.
The blackberry size can be accurately tuned by adjusting solution content and/or solution pH. The transitions between discrete macroions to blackberry structures, and between blackberries with different sizes, can be also achieved. The driving forces of the blackberry formation are not due to hydrophobic interaction, van der Waals forces or chemical interactions. Instead, we believe that the counter-ion effects and hydrogen bonds are critical. Synchrotron SAXS studies clearly show the radial distribution of small cations around large POM anions and the relation between the counter-ion association and the blackberry formation. › Continue reading
Synthesis of hybrid fluorous surfactants and their application as additives for protein renaturation
During the course of our studies with fluorous surfactants, it was discovered that fluorous hybrid detergents show promise as refolding reagents for denatured proteins. To begin to understand this phenomenon, preliminary refolding studies were performed on chemically and thermally denatured bovine carbonic anhydrase (CAB) using a series of hemifluorinated surfactants containing a sulfonate head group. Preliminary data shows that these surfactants provide good refolding yields at low as well as high concentrations of CAB compared to their hydrocarbon analogs. In contrast to traditional surfactants, we discovered that hybrid fluorous surfactants are most efficient at concentrations well below their critical micelle concentrations. This talk will present the synthesis and characterization of various hybrid fluorous surfactants and their utility in the renaturation of various structurally different proteins. The relationship between surfactant structure and efficiency in renaturation will also be discussed.
Free-Solution Comparison of DNA Hybridization using Isothermal Titration Calorimetry and Backscattering Interferometry
The characterization of biomolecular binding interactions is crucial to the understanding of biological processes and comparison of therapeutic efficacies. This study uses the novel technique of backscattering interferometry with an ITC benchmark to examine the impact of surface immobilization and the presence of a labeled probe on binding affinity. Specifically, the binding affinity of DNA hybridization with and without a fluorescent probe will be compared using both free-solution and surface-bound methodologies. The results from these experiments show a significant increase in the binding affinity for free-solution, label-free DNA hybridization compared to fluorescently labeled, surface-bound DNA hybridization. Additionally, the utility of backscattering interferometry will be compared to ITC as an analytical technique for studying various intermolecular interactions.
Positional analyses of BRCA1-dependent expression in Saccharomyces cerevisiae
Mutations in BRCA1 account for a significant proportion of hereditary breast and ovarian cancers, but analysis of BRCA1 function is complicated by pleiotropic effects and binding partners (Pol II holoenzyme and transcription factors, chromatin remodelers, recombination complexes and E3 ligases). In vertebrate cells, efforts to elucidate BRCA1 transcriptional effects have focused on specific genes or restricted portions of the genome – limiting analyses of BRCA1 effects on adjoining DNA sequences and along chromosome lengths. Here, we use microarray analyses on the genetically tractable yeast cell system to elucidate BRCA1-dependent genome-wide positional effects on both gene induction and repression. Yeast responses may be of clinical relevance based on findings that BRCA1 severely diminishes yeast growth kinetics but that BRCA1 mutated at sites identified from breast tumors is no longer able to retard yeast. Our analysis reveals that BRCA1 acts through both transcription factors to up-regulate specific loci and chromatin remodeling complexes to effect global changes in gene expression. BRCA1 also exhibits gene repression activities. Cluster-functional analysis reveals that these repressed factors are required for mitotic stability and provide a novel molecular explanation for the conditional lethality observed between BRCA1 and chromosome segregation genes.
RVS support from the Susan G. Komen for the Cure Foundation (Award BCTR0707708)
Gene Expression Profiles of HCT116-p53-/- cells Treated with Paclitaxel or Stathmin-siRNA
Among the most difficult cancers to treat are those having mutated or non-functional p53, with concurrent overexpression of stathmin (stmn1), amicrotubule (MT) destabilizer (e.g. Yuan et al. 2006. J. Pathol. 209: 549-8). Alli et al. (2007. Oncogene. 26:1003-12) demonstrated that this type of cancer cell undergoes apoptosis in response to stmn1 knock down by siRNA. The mechanism by which stmn1 siRNA promotes apoptosis in cancerous cell lines is not understood, while efficient delivery of targeted siRNA therapies remains an elusive challenge. In this work, we aim to understand the apoptotic processes activated by stmn1 siRNA in a p53-null human colon cancer cell line, HCT116-p53-null, and specifically what is unique about stmn1 siRNA induced apoptosis compared to that induced by paclitaxel treatment, a MT stabilizing drug. We find that both stmn1 depletion and paclitaxel addition slowed cell growth and caused ~25% cell death beginning ~48 h after initial treatment. Microarray analyses of gene expression profiles were then examined to provide an unbiased screen for genes up or down regulated by either treatment. Time courses (~55-72 h) were analyzed for annotated genes showing expression fold changes of greater than or equal to 2. In stmn1 siRNA treated cells, we found 215 down-regulated and 157 up-regulated genes. Paclitaxel treatment resulted in 162 down-regulated and 642 up-regulated genes. Of the differentially regulated genes from each treatment, the number of shared genes included 27 of 350 (8%) down-regulated genes and 89 of 710 (13%) up-regulated genes. These data indicate that most expression changes were unique to each treatment. Comparing those genes whose expression is oppositely changed between the two treatments also demonstrated a low level of overlap (95 of 1081 genes, or ~9%) between treatments. To date our results indicate that stmn1 siRNA and paclitaxel induce apoptosis through unique upstream signals, although each treatment acts to stabilize MTs. Continued analysis and microarray verification are ongoing to confirm our conclusions. Understanding how stmn1 siRNA induces apoptosis could lead to identification of novel chemotherapy targets.
Partially funded by an HHMI grant to Lehigh and NIH (LC).