Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells

Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear localization are either toxic or prone to crosstalk with endogenous protein cascades within live animals. We sought to take advantage of salicylic acid (SA), a small molecule that has been extensively used in humans. In plants, SA functions as a hormone that can mediate immunity and is sensed by the nonexpressor of pathogenesis-related (NPR) proteins. Although it is well recognized that nuclear translocation of NPR1 is essential to promoting immunity in plants, the exact subdomain of Arabidopsis thaliana NPR1 (AtNPR1) essential for SA-mediated nuclear translocation is controversial. Here, we utilized the fluorescent protein mCherry as the reporter to investigate the ability of SA to induce nuclear translocation of the full-length NPR1 protein or its C-terminal transactivation (TAD) domain using HEK293 cells as a heterologous system. HEK293 cells lack accessory plant proteins including NPR3/NPR4 and are thus ideally suited for studying the impact of SA-induced changes in NPR1. Our results obtained using a stable expression system show that the TAD of AtNPR1 is sufficient to enable the reversible SA-mediated nuclear translocation of mCherry. Our studies advance a basic understanding of nuclear translocation mediated by the TAD of AtNPR1 and uncover a biotechnological tool for SA-mediated nuclear localization.     

Synthetic studies of cis-4-Amino-L-proline derivatives as novel lipid lowering agents

cis-4-Amino-L-proline derivatives 1 and 2 derived from quinazolinone and pyrazolo pyrimidone respectively were designed as novel lipid lowering agents. A preliminary in vivo screening indicates that 1b and 2a have moderate triglyceride lowering activity.     

Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells

The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).     

Molecular cloning,characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.     

Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells

Background

Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions.

Methods

We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone.

Results

Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells.

Conclusions

Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.     

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.     

Circadian dysfunction reduces lifespan in Drosophila melanogaster

Circadian clocks regulate physiological and behavioral processes in a wide variety of organisms, and any malfunction in these clocks can cause significant health problems. In this paper, we report the results of our study on the physiological consequences of circadian dysfunction (malfunctioning of circadian clocks) in two wild-type populations of fruit flies (Drosophila melanogaster). We assayed locomotor activity behavior and lifespan among adult flies kept under constant dark (DD) conditions of the laboratory, wherein they were categorized as rhythmic if their activity/rest schedules followed circadian (approximately 24 h) patterns, and as arrhythmic if their activity/rest schedules did not display any pattern. The rhythmic flies from both populations lived significantly longer than the arrhythmic ones. Based on these results, we conclude that circadian dysfunction is deleterious, and proper functioning of circadian clocks is essential for the physiological well being of D. melanogaster.