Thermodynamic effects of phospholamban on Ca-ATPase kinetics

The Ca-ATPase of sarcoplasmic reticulum removes cytosolic calcium to promote muscle relaxation. In the heart, the Ca-ATPase is regulated by phospholamban, which inhibits the Ca-ATPase by decreasing Ca-ATPase calcium sensitivity. However, the kinetic and thermodynamic mechanisms of inhibition are not understood. The purpose of this research was to test the hypothesis that phospholamban regulates Ca-ATPase kinetics by increasing Ca-ATPase activation energy. The baculovirus-insect cell expression system was used to produce samples containing Ca-ATPase alone or Ca-ATPase with phospholamban. The temperature-dependence of Ca-ATPase activity and catalytic site density was measured in the absence and presence of phospholamban at sub-saturating calcium and used to calculate the temperature-dependence of Ca-ATPase turnover. Arrhenius analyses showed that phospholamban increased Ca-ATPase activation energy from 31 +/- 3 J/mol (Ca-ATPase only) to 52 +/- 5 J/mol (Ca-ATPase + phospholamban). The results supported the hypothesis and provided new insight into the mechanism of phospholamban inhibition of Ca-ATPase

Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling

<p>Abstract</p> <p>Background</p> <p>Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human.</p> <p>Results</p> <p>The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS) and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery.</p> <p>Conclusion</p> <p>Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.</p

Application of Magnetic Nanoparticles in Pharmaceutical Sciences

# The Author(s) 2010. This article is published with open access at Springerlink.com KEY WORDS magnetic beads. magnetic bioseparation. magnetic nanoparticle

An approach to the identification of trypanosoma congolense chromosome-specific hybridization markers.

Chromosome rearrangements that occur in trypanosomes appear to affect mainly the medium-sized chromosomes. A possibility exists that non-duplicative activation of telomeric VSG genes might involve chromosome rearrangements occurring several kilobase pairs upstream of the affected VSG gene, thus remaining undetectable by standard gel electrophoresis and southern blot hybridizations with VSG gene probes. Since trypanosomes do not condense their chromosomes at any stage of cell division, this hypothesis cannot be tested by cytological hybridization. Some of the size alterations in chromosomes occur when the trypanosomes are repeatedly passaged, cyclically transmitted through tsetse flies or grown in rodents in the presence of trypanocidal drugs. In some instances, the alterations in the sizes of some chromosomes may relate to a changed phenotype of the parasite. Pulsed field gradient gel electrophoresis (PFGE) of chromosome-sized DNA molecules of lower eukaryotic organisms is an effective way of characterizing such organisms. It has been used to characterize many different isolates of parasitic protozoa including Plasmodium falciparum, Leishmania spp., Trypanosoma spp., Theileria spp. and Toxoplasma. When a particular chromosome rearranges in such organisms, it is impossible to identify the rearranged chromosome in the absence of a marker specific for the chromosome. Short oligonucleotide primers (1O-mers) of arbitrary nucleotide sequences have been used in the polymerase chain reaction (PCR) to generate genomic fingerprints that facilitate the characterization and differentiation of various organisms and for physical mapping of loci which contain genes responsible for identifiable phenotypes. It was proposed that the use of such primers to amplify purified individual chromosomes may generate fingerprints which are characteristic of each chromosome. To test this hypothesis, T congolense clone IL 1180 was used because of its sensitivity to trypanocides and clear pedigree. PFGE conditions were optimized which best separates the four medium-sized chromosomes, whose sizes are: 340 kb, 360 kb, 400 kb and 500 kb. The four chromosomes thus separated were individually purified from agarose gel slices and then peR-amplified with the random primers. The fingerprints of DNA fragments generated were resolved by standard agarose gel electrophoresis. The majority of primers used generated reproducible fingerprints, some of which were polymorphic for the different chromosomes used. In this way, a fragment of DNA was identified which hybridizes only to one of the four chromosomes from which it was amplified by a random primer. Thus, taking this approach, it is feasible to generate chromosome-specific hybridization markers

Molecular mechanisms of nuclear factor-erythroid-2 related factor 2 (Nrf2) regulation: Phosphorylation by casein kinase 2 (CK2) and interaction with proto -oncogene N -Myc in neuroblastoma cells.

Environmental pollutants and toxicants expose cells to oxidative and electrophilic challenges. Oxidative and xenobiotic stresses caused by environmental toxicants are known to cause human diseases including cancer and diabetes. To protect against the insults, cells have developed detection and response mechanisms. One such sensor-defense cellular mechanism involves the Nrf2 pathway. The transcription factor Nuclear factor erythriod 2 related factor 2 (Nrf2), a member of the cnc bZIP transcription factors, regulates the expression and induction of phase II antioxidant and detoxification genes via antioxidant response element (ARE). In the presence of an antioxidant, Nrf2 has been shown to be activated, translocate into the nucleus upon activation, heterodimerize with small Maf proteins like MafG and K, then bind to the ARE and activate transcription of phase II genes. Nrf2 has been shown to be important in neuronal function since ablation of Nrf2 induces leukoencephalopathy with wide spread gliosis in mouse brain. The signaling events leading to the activation of Nrf2 has not been clear; in particular, the role of phosphorylation in Nrf2 function remains controversial. We report that phenolic compounds, like antioxidant tert-butylhydroquinone (tBHQ) induced two forms of the Nrf2 protein in neuroblastoma cells (IMR-32), which migrated as distinctive bands on SDS-PAGE. The two bands were characterized to consist of the phosphorylated and the non phosphorylated forms of Nrf2. In vitro treatment with λ phosphatase eliminated the slower migrating form and increased the amount of the faster migrating form of Nrf2. While unphosphorylated Nrf2 predominated in the cytoplasm, the phosphorylated form preferentially localized in the nucleus. Deletional analyses from both the carboxyl- and the amino-ends revealed the transcription activation (TA) domains Neh4 and Neh5 as a major region necessary for the phosphorylation. The TA domains were characterized by the presence of multiple conserved phosphorylation sites of casein kinase 2 (CK2). Treatments with CK2 inhibitor DMAT blocked the induction of endogenous target genes of Nrf2 in cells and also significantly inhibited the TA activities of both the full length and the TA domains of Nrf2. Furthermore, phosphorylation of the TA domains correlated with the nuclear translocation of Nrf2 that was inhibited by DMAT in a concentration dependent manner. The findings demonstrated that phosphorylation of Nrf2 at the TA domains by CK2 is an integral component of Nrf2 activation necessary for the nuclear localization and transcription activation function of Nrf2 in neuroblastoma cells. Nrf2 activity is significantly affected by its interacting partners. I intended to identify new Nrf2 interacting partners. Using the yeast 2-hybrid assay, I identified N-Myc as a novel Nrf2-interacting protein. In addition, I identified MafG and ATF4, which have been known to interact with and affect Nrf2, and several other new proteins interacting with Nrf2. N-Myc is a known proto-oncogene required for embryonic development and organogenesis. Over expression of N-Myc leads to aggressive neuroblastomas. Further characterization revealed that N-Myc interacts with the C-terminal region of Nrf2. The C-terminal region of Nrf2 is involved in DNA binding and heterodimerization and therefore interaction of this region with N-Myc implicate the interaction in the transcriptional regulation of Nrf2 target gene and in cellular survival

Trypanosoma (nannomonas) congolense: separation of chromosomes of PCR product specific to medium-size chromosome

Pulsed field gradient gel electrophoresis was used to separate chromosomes of parasitic protozoan Trypanosome congolense clone IL1180. Total trypanosome DNA was isolated and resolved into 18 chromosomes by pulsed-field gradient gel electrophoresis. The chromosomes fall into four main size categories: the minichromosomes of about 50-150 kb: the medium-size chromosomes 200-750kb; the large chromosomes>1mb and chromosomes that are –specifically trapped in the wells. Four distinct medium –sized chromosomes with sizes 340kb, 360kb, 400kb and 500kb, were identified and designated chromosomes 1-4 respectively. DNA bands from each of the four medium-size chromosomes were excised from the gel, purified and randomly amplified with 23 random oligonucleotide primers (RAPD analysis). The RAPD product, which was unique to each of the four medium –sized chromosomes, were identified and cloned in T-vector plasmid. One RAPD product of approximate size 1.5Kb, which was amplified by oligonucleotide primer, ILO867 was identified as unique to medium –sized chromosome2. This RAPD product referred to as RAPD2/867/1.5 hereinafter only hybridized to medium sized chromosomes2. This cloned fragment can therefore be used as a specific DNA marker for medium –size chromosome 2 of T. congolense. The Kenya Veterinarian Vol. 27 2004: pp. 91-9

Accelerated Ovarian Failure Induced by 4-Vinyl Cyclohexene Diepoxide in Nrf2 Null Mice

Genetic and biochemical analyses have uncovered an essential role for nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating phase II xenobiotic metabolism and antioxidant response. Here we show that Nrf2 protects against the ovarian toxicity of 4-vinylcyclohexene diepoxide (VCD) in mice. Nrf2(−/−) female mice exposed to VCD exhibit an age-dependent decline in reproduction leading to secondary infertility accompanied by hypergonadotropic hypogonadism after 30 weeks of age. VCD is shown to selectively destroy small ovarian follicles, resulting in early depletion of functional follicles. Treatment with VCD induces apoptotic death in cultured cells and in ovarian follicles, suggesting apoptosis as a mechanism of follicle loss. Loss of Nrf2 function blocks the basal and inducible expression of microsomal epoxide hydrolase, a key enzyme in the detoxification of VCD, and increases the oxidative stress in cells that is further exacerbated by VCD. Foxo3a, a repressor in the early stages of follicle activation, displays reduced expression in Nrf2(−/−) ovaries, causing accelerated growth of follicles in the absence of exposure to exogenous chemicals. Furthermore, Foxo3a is degraded through the 26S proteasome pathway in untreated cells and is induced by VCD via both Nrf2-dependent transcription and protein stabilization. This study demonstrates that Nrf2 serves as an essential sensor and regulator of chemical homeostasis in ovarian cells, protecting the cells from toxic chemicals by controlling metabolic detoxification, reactive oxygen species defense, and Foxo3a expression. In addition, these findings raise the possibility that exposure to environmental or occupational ovotoxicants plays a role in the premature ovarian failure commonly associated with infertility and premature aging in women