Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites

The formation of protein aggregates (foci) at sites of DNA double-strand breaks (DSBs) is mainly studied by immunostaining and is hence limited by the low resolution of light microscopy and the availability of appropriate and selective antibodies. Here, we describe a system using enzymatic creation of site-specific DNA DSBs within the human genome combined with chromatin immunoprecipitation (ChIP) that enables molecular probing of a DSB. Following induction of the I-PpoI enzyme and generation of DSBs, cellular DNA and proteins are crosslinked and analyzed by ChIP for specific proteins at the site of the break. The system allows the direct detection of protein and chromatin dynamics at the site of the break with high resolution, as well as direct measurement of DNA repair defects in human cells. Starting with fragmented chromatin, results can be achieved in 2-3 d.     

Effects of β-adrenoceptor blocker pindolol, calcium antagonist verapamil and their combination on retention in step-down- and shuttle-box-trained rats and on brain biogenic monoamines

The effects of the β-adrenoceptor blocker pindolol and the calcium antagonist verapamil administered alone or in combination on retention in step-down- and shuttle-box-trained rats and on the biogenic monoamine levels in the frontal cortex and hippocampus were examined. The chronic oral treatment with pindolol impaired retention in step-down- and shuttle-box-trained rats, decreasing the dopamine (DA) and noradrenaline (NA) levels and increasing the serotonin (5-HT) levels in the cortex and hippocampus. Verapamil did not influence retention in step-down- and shuttle-box avoidance situation and the biogenic monoamine levels in the frontal cortex and hippocampus. It should, however, be noted that the chronic oral treatment with verapamil completely abolished the retention-impairing effect of pindolol, restoring to normal DA, NA and 5-HT levels. These findings might be of interest to clinical practice and suggest the necessity for using a combination of β-blockers with Ca²⁺ antagonists in case of prolonged treatment of cardiovascular diseases.     

Pregnancy success of lactating Holstein cows after a single administration of a sustained-release formulation of recombinant bovine somatotropin

Background

Results regarding the use of bovine somatotropin for enhancing fertility in dairy cattle are variable. Here, the hypothesis was tested that a single injection of a sustained-release preparation of bovine somatotropin (bST) during the preovulatory period would improve pregnancy success of lactating dairy cows at first service.

Results

The first experiment was conducted in a temperate region of Mexico. Cows inseminated following natural estrus or timed artificial insemination were given a single injection of bST or a placebo injection at insemination (n = 100 cows per group). There was no significant difference between bST and control groups in the proportion of inseminated cows diagnosed pregnant (29 vs 31% pregnant). The second experiment was performed during heat stress in Florida. Cows were subjected to an ovulation synchronization regimen for first insemination. Cows treated with bST received a single injection at 3 days before insemination. Controls received no additional treatment. As expected, bST did not increase vaginal temperature. Treatment with bST did not significantly increase the proportion of inseminated cows diagnosed pregnant although it was numerically greater for the bST group (24.2% vs 17.8%, 124–132 cows per group). There was a tendency (p = 0.10) for a smaller percent of control cows to have high plasma progesterone concentrations (≥ 1 ng/ml) at Day 7 after insemination than for bST-treated cows (72.6 vs 81.1%). When only cows that were successfully synchronized were considered, the magnitude of the absolute difference in the percentage of inseminated cows that were diagnosed pregnant between bST and control cows was reduced (24.8 vs 22.4% pregnant for bST and control).

Conclusion

Results failed to indicate a beneficial effect of bST treatment on fertility of lactating dairy cows.

     

Expression of γ-H2AX and patient prognosis in breast cancer cohort

H2AX phosphorylation is a novel marker of DNA double-stranded breaks. In the present study, we assessed the γ-H2AX expression, its association with other clinicopathologic characteristics, and the prognosis in a cohort of 97 patients with breast cancer. Ninety-seven specimens of tumor tissue and 77 adjacent normal tissues from patients with breast cancer were examined. All patients underwent modified radical mastectomy or local tumor resection without lymph node dissection. γ-H2AX expression was assessed by standard immunohistochemistry. Patients were followed after surgery for a mean duration of 70.1 ± 18.7 months (range, 6-93 months). The γ-H2AX staining was positive in 27 (27.8%) patients. The positive rates of H2AX were 26.0% and 2.6% in tumor tissue and adjacent normal tissues, respectively. γ-H2AX positive status was negatively associated with TNM staging, with 24 positive cases (32.4%) in TNM staging I-II, while no positive cases in TNM staging III-IV (P = 0.026). Sixteen patients (16.5%) died during the follow-up. No significant association between γ-H2AX expression and patient survival was detected. The unadjusted HR (hazard ratio) for γ-H2AX positive was 0.84 (95% CI: 0.27, 2.60). In TNM staging subgroup analysis, death only occurred in γ-H2AX negative patients. Our study is the first study to demonstrate that expression of γ-H2AX is associated with TNM staging. Due to the small sample and limited follow-up time, we did not observe a significant association between γ-H2AX and patient survival. γ-H2AX expression could be a potential biomarker for cancer diagnosis and prediction, and further studies are in need.     

Posters

Introduction  Fine needle aspiration (FNA) cytology of the thyroid is a well-established test in the clinical work-up of patients with solitary nodules of the thyroid. Thyroid FNA does however have limitations and audit of diagnostic performance is important.
Methods  The histopathology archives of the Royal Victoria Hospital were searched for all thyroid resections and the histopathological diagnosis was correlated with the pre-operative cytological diagnosis, where available. Special emphasis was placed on the accuracy of tumour diagnosis.
Results  A total of 173 cases were identified during the 2-year period, of these 93 had available pre-operative FNA. A total of 57 tumours were identified. A small number (six of 57) of significant discrepancies were identified. These included a malignant lymphoma diagnosed as Hashimoto's thyroiditis, a metastasis which the FNA had suggested was a medullary carcinoma and an insular carcinoma diagnosed as medullary carcinoma on FNA. False positives included a colloid cyst diagnosed as suspicious of malignancy and a cytological diagnosis of papillary carcinoma not confirmed on histology.
Discussion  At present, the majority of thyroid FNAs in our clinics are performed by surgeons and material is not routinely available for immunocytochemistry. In spite of these limitations, there were few major discrepancies. These might be reduced if pathologist aspirators were able to perform FNAs and collect material for further studies, where necessary. This would allow identification of medullary carcinomas and malignant lymphomas.
Conclusion  FNA of thyroid lesions is a useful investigation in our clinical setting, however, some areas of potential for improvement have been identified.     

Parc-ing p53 in the cytoplasm

Nikolaev et al. (this issue of Cell) report the identification of a parkin-like protein, Parc, and its role in anchoring the tumor suppressor protein p53 in the cytoplasm reveals yet another level of control of p53 function. Regulation of the subcellular localization of p53 by Parc may serve as a novel target in treatment of certain types of tumors.     

Chromatin association of rad17 is required for an ataxia telangiectasia and rad-related kinase-mediated S-phase checkpoint in response to low-dose ultraviolet radiation

Activation of the S-phase checkpoint results in an inhibition of DNA synthesis in response to DNA damage. This is an active cellular response that may enhance cell survival and limit heritable genetic abnormalities. While much attention has been paid to elucidating signal transduction pathways regulating the ionizing radiation-induced S-phase checkpoint, less is known about whether UV radiation initiates the process and the mechanism controlling it. Here, we demonstrate that low-dose UV radiation activates an S-phase checkpoint that requires the ataxia telangiectasia and Rad-related kinase (ATR). ATR regulates the S-phase checkpoint through phosphorylation of the downstream target structural maintenance of chromosomal protein 1. Furthermore, the ATPase activity of Rad17 is crucial for its chromatin association and for the functional effects of ATR activation in response to low-dose UV radiation. These results suggest that low-dose UV radiation activates an S-phase checkpoint requiring ATR-mediated signal transduction pathway.