Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice

Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3(-/-) mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3(-/-) mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3(-/-) mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3(-/-) mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3(+/+) cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3(-/-) and Hsp70.1/3(+/+) fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3(-/-) cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3(-/-) cells in comparison to Hsp70.1/3(+/+) cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.     

HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts

We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(-/-)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(-/-) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(-/-) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.     

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.     

Pyrazole compound BPR1P0034 with potent and selective anti-influenza virus activity

Background

Influenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern. Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals.

Methods

We searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay. After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques. Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle.

Results

The 50% effective inhibitory concentration (IC₅₀) of BPR1P0034 was 0.42 ± 0.11 μM, when measured with a plaque reduction assay. Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9. In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus.

Conclusions

To the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-μM) antiviral activity. We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.     

Transformation of potato via Agrobacterium coated microparticle bombardment

The transformation of potato (Solanum tuberosum L. cv. Désirée) was extended by the Agrobacterium-mediated biolistic method. Using this approach transgenic shoots could be obtained at a similar frequency to that achieved through conventional biolistics. Leaves from shoot cultures were bombarded with gold particles coated in Agrobacterium tumefaciens cells harboring a binary plasmid encoding three genes of interest in the T-DNA. Nine shoots were obtained from 20 shots, with selection of transgenic shoots on a series of media containing progressively increasing concentrations of hygromycin from 5 to 20 mg dm⁻³.     

KUD773, a phenylthiazole derivative,displays anticancer activity in human hormone-refractory prostate cancers through inhibition of tubulin polymerization and anti-Aurora A activity

Background

Hormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.

Results

KUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr¹⁶¹ (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr¹⁵ (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.

Conclusions

The data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs.