Functionally redundant isoforms of a yeast Hsp70 chaperone subfamily have different antiprion effects

Why eukaryotes encode multiple Hsp70 isoforms is unclear. Saccharomyces cerevisiae Ssa1p and Ssa2p are constitutive 98% identical Hsp70's. Stress-inducible Ssa3p and Ssa4p are 80% identical to Ssa1/2p. We show Ssa1p-4p have distinct functions affecting [PSI(+)] and [URE3] prions. When expressed as the only Ssa, Ssa1p antagonized [URE3] and Ssa2p antagonized [PSI(+)]. Ssa3p and Ssa4p influenced [URE3] and [PSI(+)] somewhat differently but overall their effects paralleled those of Ssa1p and Ssa2p, respectively. Additionally, Ssa3p suppressed a prion-inhibitory effect of elevated temperature. Our previously described Ssa1-21p mutant weakens [PSI(+)] in SSA1-21 SSA2 cells and abolishes it in SSA1-21 ssa2Delta cells. To test if the same mutation affected other prions or altered Ssa2p similarly, we compared effects of a constructed Ssa2-21p mutant and Ssa1-21p on both prions. Surprisingly, [URE3] was unaffected in SSA1-21 SSA2 cells and could propagate in SSA1-21 ssa2Delta cells. Ssa2-21p impaired [URE3] considerably and weakened [PSI(+)] strongly but in a manner distinct from Ssa1-21p, highlighting functional differences between these nearly identical Hsp70's. Our data uncover exquisite functional differences among isoforms of a highly homologous cytosolic Hsp70 subfamily and point to a possibility that variations in Hsp70 function that might improve fitness under optimal conditions are also important during stress.     

Non-redundant roles for Profilin2 and Profilin1 during vertebrate gastrulation

Gastrulation is a critical morphogenetic event during vertebrate embryogenesis, and it is comprised of directional cell movement resulting from the polarization and reorganization of the actin cytoskeleton. The non-canonical Wnt signaling pathway has emerged as a key regulator of gastrulation. However, the molecular mechanisms by which the Wnt pathway mediates changes to the cellular actin cytoskeleton remains poorly defined. We had previously identified the Formin protein Daam1 and an effector molecule XProfilin1 as links for Wnt-mediated cytoskeletal changes during gastrulation. We report here the identification of XProfilin2 as a non-redundant and distinct effector of Daam1 for gastrulation. XProfilin2 interacts with FH1 domain of Daam1 and temporally interacts with Daam1 during gastrulation. In the Xenopus embryo, XProfilin2 is temporally expressed throughout embryogenesis and it is spatially expressed in cells undergoing morphogenetic movement during gastrulation. While we have previously shown XProfilin1 regulates blastopore closure, overexpression or depletion of XProfilin2 specifically affects convergent extension movement independent of mesodermal specification. Specifically, we show that XProfilin2 modulates cell polarization and axial alignment of mesodermal cells undergoing gastrulation independent of XProfilin1. Together, our studies demonstrate that XProfilin2 and XProfilin1 are non-redundant effectors for Daam1 for non-canonical Wnt signaling and that they regulate distinct functions during vertebrate gastrulation.     

Structure–activity relationship studies of curcumin analogues

Two series of curcumin analogues, a total of twenty-four compounds, were synthesized and evaluated. The most potent compound, compound 23, showed potent growth inhibitory activities on both prostate and breast cancer lines with IC₅₀ values in sub-micromolar range, fifty times more potent than curcumin. Curcumin analogues might be potential anti-tumor agents for breast and prostate cancers.     

Tobacco carcinogen induces microglial activation and subsequent neuronal damage

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific procarcinogen. We have investigated whether NNK causes inflammatory upheaval in the brain by activation of resident microglia and astrocyte and result in bystander neuronal damage. We have carried out the work in both in vitro and in vivo models. We have found that treatment with NNK causes significant activation of mouse microglial (BV2) cell line as evident by increase in reactive oxygen species and nitric oxide level. Western blot analysis has showed increase in proinflammatory signaling proteins, proinflammatory effector proteins, and other stress-related proteins. Interestingly, increased levels of proinflammatory cytokines like interleukin (IL)-6, tumor necrosis factor-α, monocyte chemoattractant protein 1 (MCP1), and IL-12p70 are also detected. Work from our in vivo studies has demonstrated similar increase in proinflammatory signaling and effector molecules along with the proinflammatory cytokine levels, following NNK treatment. Immunohistochemical staining of the brain sections of NNK-treated mice reveals massive microglial and astrocyte activation along with distinct foci of neuronal damage. Both in vitro and in vivo results provide strong indication that NNK causes significant upheaval of the inflammatory condition of brain and inflicts subsequent neuronal damage.     

Identification and characterization of a novel amphioxus dopamine D1-like receptor

Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D₁ and D₂) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus ( Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D₁-like receptor sequences. One of these receptors, AmphiD₁/β, was found to have high levels of sequence similarity to both vertebrate D₁ receptors and to β-adrenergic receptors. Here, we report on the cloning of AmphiD₁/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD₁/β has a similar pharmacology to vertebrate D₁ receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits 'agonist-specific coupling' depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD₁/β may be an amphioxus orthologue of vertebrate β-adrenergic receptors.