Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

Short hairpin RNAs against eotaxin or interleukin‐5 decrease airway eosinophilia and hyper‐responsiveness in a murine model of asthma

Background

Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up‐regulation of eotaxin, which, together with interleukin (IL)‐5, is important for differentiation, chemo‐attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus‐delivered short hairpin RNA (shRNA) to suppress the expression of IL‐5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL‐5 shRNAs on airway inflammation in an ovalbumin (OVA)‐induced murine model of asthma.

Methods

Lentivirus‐delivered shRNAs were used to suppress the expression of eotaxin and/or IL‐5 in local tissue in an OVA‐induced murine asthma model.

Results

Intra‐tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper‐responsiveness, cellular infiltration of lung tissues, and eotaxin and IL‐5 levels in bronchio‐alveolar lavage fluid. Administration of lentiviruses expressing IL‐5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model.

Conclusions

Local delivery of lentiviruses expressing IL‐5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia. Copyright © 2008 John Wiley & Sons, Ltd.     

Finding the mechanism of esterase D activation by a small molecule

People with reduced esterase D (ESD) activity are susceptible to many diseases. However, how to activate ESD is still unknown. To address the question, we identified that 4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4, 5-dihydro-1H-pyrazol-3-yl) phenol (FPD5) could be a good candidate activator for ESD activity. We found that FPD5 could increase ESD activity in a dose-dependent way. FPD5 bound directly to ESD at Lys180 rather than its ubiquitination site Lys213. Site-directed mutagenesis at the binding site or the ubiquitination site inhibited FPD5 action. FPD5 increased the level of ESD mono-ubiquitination and mutESD K213A completely inhibited this action. Our findings highlighted the activation mechanism of ESD via promoting the mono-ubiquitination of ESD.     

Hybrid molecules based on fullerene C60 and 5Z,9Z-dienoic acids: Synthesis and cytotoxic activity

The present research project details synthesis of new hybrid methanofullerenes based on acetylene and triazole esters of malonic acid containing 5Z,9Z-dienoic acids and fullerene C₆₀ under Bingel-Hirsch conditions, including study of the cytotoxic activity with respect to Jurkat, K562, U937 and HL60 tumor cell lines. Hybrid methanofullerenes containing acetylenic fragments, unlike triazole substituents, were found to exhibit higher cytotoxicity, but are characterized by lower selectivity of action in relation to healthy cells.