Analysis of the blocking activity of charybdotoxin homologs and iodinated derivatives against Ca2+-Activated K+ channels

Summary Two charybdotoxin peptides were purified from venom of the Israeli scorpion,Leiurus quinquestriatus hebraeus. Microsequencing of the most abundant toxin, ChTX-Lq1, revealed identity with the 37-residue peptide previously sequenced by Gimenez-Gallego et al. [Gimenez-Gallego, G., et al.,Proc. Natl. Acad. Sci. USA 85:3329–3333 (1988)]. Sequence data on the minor peptide, ChTX-Lq2, showed substantial homology to ChTX-Lq1 with differences observed at eight positions. These two charybdotoxin sequences, along with that of noxiustoxin, define a distinct family of scorpion peptide toxins with activity against K⁺ channels. Both charybdotoxin homologs inhibited Ca²⁺-dependent K⁺ efflux from human erythrocytes with similar potency,K _0.5-40nm. In planar bilayer assays of single K(Ca) channels from rat muscle, ChTX-Lq1 and ChTX-Lq2 blocked with intrinsicK _d's of 1.3 and 43nm, respectively, in the presence of 50mm external KCl. A new application of dwell-time histogram analysis of single-channel blocking events was used to characterize the kinetic homogeneity of toxin samples and the blocking kinetics of ChTX derivatives. The lower blocking affinity of ChTX-Lq2 was the combined result of a faster dissociation rate and a slower association rate as compared to ChTX-Lq1. The blocking activity of two mono-iodinated derivatives of ChTX-Lq1 was also analyzed. Blocked dwell-time histograms of the iodinated peptides were characterized by predominately brief (0.2–2 sec) blocking events in comparison to the native toxin (20 sec). Histogram analysis revealed that mono-iodination of ChTX-Lq1 impairs blocking activity by adverse effects on both dissociation and association rate constants. Frequency density histograms of single channel blocking events provide a sensitive assay of toxin purity suitable for quantitating structure-activity relationships of charybdotoxin derivatives.     

Expression and distribution of grp-78/bip in mineralizing tissues and mesenchymal cells

Glucose-regulated protein 78 (GRP-78) is one of the many endoplasmic reticulum chaperone proteins that have been shown to possess multifunctional roles. We have previously demonstrated that GRP-78 functions as a receptor for dentin matrix protein 1 (DMP1) and is required for DMP1-mediated calcium release; that it is a secreted protein and can bind to type I collagen and DMP1 extracellularly and aid in the nucleation of calcium phosphate. We provide evidence in this study that tyrosine phosphorylation is required for DMP1/GRP-78-mediated calcium release in mesenchymal cells. We further demonstrate that GRP-78 is localized in the nucleus of mesenchymal cells and that the cell surface GRP-78 is not associated with the G-protein Gαq in mesenchymal cells. Results from this study show that during development of mineralized tissues, increased expression of GRP-78 can be observed in condensing cartilage and mesenchymal cells of the alveolar bone, endochondral bone and dental pulp. Additionally, we show that GRP-78 is present in the mineralizing matrices of teeth, bone and in the extracellular matrix of differentiating human marrow stromal cells and dental pulp stem cells. Collectively, our observations provide a new perspective on GRP-78 with respect to mineralized matrix formation.     

Acaricidal activity of Cassia alata against Rhipicephalus (Boophilus) annulatus

Using adult immersion test, the acaricidal activity of ethanolic extracts of leaves of Cassia alata L. was studied against Rhipicephalus (Boophilus) annulatus. The efficacy was assessed by measuring per cent adult mortality, inhibition of fecundity and hatching rate. The ethanolic extract of C. alata produced a concentration dependant increase in the adult tick mortality. The highest mortality (45.8%) and inhibition of fecundity (10.9%) were observed at the highest concentration tested (100 mg/ml). The plant extract did not affect egg hatchability.     

Translesion DNA polymerases Pol ζ, Pol η, Pol ι, Pol κ and Rev1 are not essential for repeat-induced point mutation inNeurospora crassa

Pol ζ, Pol η, Pol ι, Pol κ and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) inNeurospora crassa. Additionally, we demonstrate that theupr-1 gene, which encodes the catalytic subunit of Pol ζ, is a highly polymorphic locus in Neurospora. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession Nos DQ 231523, DQ 231524, DQ 235021, DQ 235525-DQ 235541, DQ 240287, DQ 240288, DQ 354228, DQ 354235-DQ 354237, DQ 386416-DQ 386422, DQ 387872, DQ494492-DQ494503 and DQ417211-DQ417220.     

Curcumin derivative 1, 2-bis [(3E, 5E)-3, 5-bis [(2-chlorophenyl) methylene]-4-oxo-1-piperidyl] ethane-1, 2-dione (ST03) induces mitochondria mediated apoptosis in ovarian cancer cells and inhibits tumor progression in EAC mouse model

Curcumin is known for its anticancer properties, but its clinical application is limited due to its poor bioavailability and chemical stability. In this study we report the curcumin derivative, ST03 (1,2-bis[(3E,5E)-3,5-bis[(2-chlorophenyl)methylene]-4-oxo-1-piperidyl]ethane-1,2-dione) exhibits ∼ 14 fold better bioavailability compared to curcumin and is detectable in plasma up to 12 h. ST03 induces ROS, activates the intrinsic apoptotic pathway as evident by disruption of mitochondrial membrane potential, and induction of proapoptotic proteins in ovarian cancer lines PA1 and A2780. ST03 also blocked the migration of ovarian cancer cells. ST03 exerted its antitumor effect in-vivo in the EAC mouse model by activating the intrinsic apoptotic pathway. Our findings demonstrate ST03, a curcumin derivative, with better bioavailability and stability with no discernable toxicity in vivo to be a promising drug candidate for anticancer therapies.