Caffeine effect on the mitotic delay induced by G2 treatment with UVC or mitomycin C

It is well established that DNA lesions trigger cell cycle check-pointscausing a mitotic delay that is required for their repair beforecells enter the mitotic phase. Caffeine, in some cases, canremove this delay and consequently potentiates the yield ofinduced chromosome aberrations. The objective of this studywas to test the effect of a G2 treatment with S-dependent agents(UV light and mitomycin C) on the cell kinetics of a G2 cellpopulation and evaluate whether post-treatments with caffeinecould modulate removal of the expected cell cycle delay. Cellkinetics were monitored by analysing the mitotic index (MI)values in combination with the 5-bromo-2'-deoxyuridine (BrdUrd)labelling technique. Chinese hamster fibroblast cultures (AA8)were treated in G2 phase of the cell cycle with 8 and 15 J/m²UV light or 0.1 and 0.6 µg/ml mitomycin C for 1.5 h. Post-treatmentswith caffeine were performed at dose levels and recovery timeswhere the mitotic indices were substantially reduced. The resultsobtained showed that both UV light and mitomycin C induced aG2 arrest, as indicated by MI values and the absence of Brd-Urdlabelledmetaphases. For UV light the G2 block was observed at lowerand higher dose levels after 1.5 h, while for mitomycin C itwas observed only at the higher dose level after 1 h. However,in both cases the block lasted {small tilde}1 h, after which,even though slowed down, the cell population entered mitosis,as indicated by increased MI values. This block was not removedby caffeine post-treatment. In contrast, caffeine G2 post-treatmentwas able to remove G2 arrest induced by G1–treatments.Accordingly, our results suggest that both UV light- and mitomycinC-induced damage must be processed during S phase to allow caffeineto remove induced G2 blocks.      

Distribution of camptothecin-induced break points in Chinese hamster cells treated in late S and G2 phases of the cell cycle

The distribution of camptothecin (CPT)-induced break pointsin late S or G2 phase of the cell cycle observed in Chinesehamster chromosomes was analysed in 400 metaphases. Contraryto expectation, they were not localized in the heterochromaticregions, suggesting that these chromatid-type aberrations ariseby a mechanism which does not involve collision of the CPT-trapped'cleavable complex' with the replication fork. Since many breakpoints mapped more frequently to light bands (DAPI negative)than dark bands (DAPI positive) with a frequency of 73 and 15%respectively, it could be argued that the presence of the CPT-trapped'cleavable complex' probably interferes with chromatin condensation.In fact, the euchromatic regions, which are expected to be moreactively condensed in G₂ phase, were more involved in chromosomaldamage. These results do not completely confirm the idea thatsome residual DNA synthesis occurring in G₂is responsible forthe G₂ clastogenic effects of CPT as the heterochromatic regionsshould, in this case, be more involved. ³To whom correspondence should be addressed. Tel: +39 761 357 206; Fax: +39 761 357 242; Email: palitti{at}unitus.it     

Catalytic inhibition of topoisomerase II in Werner's syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of the cell cycle

PURPOSE: To investigate whether catalytic topoisomerase II activity by ICRF187, a compound that interferes with the catalytic cycle of topoisomerase II without causing DNA damage, could result in a modulation of X-ray-induced chromosomal damage in Werner's syndrome (WS) cell lines. MATERIALS AND METHODS: Two WS (KO375, DJG) and one normal lymphoblastoid cell line (SNW646) were exposed to X-rays, post-treated with ICRF187 and harvested after various recovery times. Cell progression to mitosis was monitored by 5-bromo-2'-deoxyuridine (BrdUrd) and fluorescent immmunodetection to analyse chromosomal damage in homogeneous treated cell populations in the G1, S or G2 phase of the cell cycle. RESULTS: In WS cell lines, catalytic inhibition of topoisomerase II activity by ICRF187 resulted in potentiation of X-ray- induced chromosomal damage in the G2 phase of the cell cycle. This potentiation was not observed in the G1 or S phases of the cell cycle, neither in WS nor normal cells. CONCLUSION: These results point out the possibility that Werner's syndrome protein (WRNp) might play a role in a G2 recombinational pathway of double-strand break repair, cooperating with topoisomerase II and thus contributing to maintain genomic integrity.     

Serum Ornithine Carbamoyl Transferase as a Surrogate Marker in Malaria

Ornithine carbamoyl transferase (OCT) activity and other liver function tests were studied in a total of 50 patients of clinical malaria and 15 controls. They were grouped as group I (positive for malarial parasite on peripheral blood smear, n=18), group II (negative for malarial parasite on peripheral blood smear (PBS) but responded to antimalarials, n=17) and group III (peripheral blood smear negative and did not respond to antimalarial therapy, n=15). The mean OCT levels were significantly raised in group I (6.79 ± 1.84 IU/L, p value = 0.006) and group II (5.0 ± 1.15 IU/L, p value = 0.014) as compared to controls (2.5 ± 1.13 IU/L) and returned to normal after treatment In contrast, group III had normal levels except in a case of kala azar and septicemia where OCT levels were high and increased further on treatment. Taking PBS positivity as a gold standard of diagnostic criteria, OCT had a sensitivity of 83% and specificity of 86% with a high positive predictive value of 88% as compared to ALT which had a lower sensitivity of 55% and specificity of 80%. The clinical response rate in PBS negative cases of fever having high OCT level was 83% as compared to 35% in cases with normal OCT level, making OCT a good surrogate marker of malaria. OCT levels could also be of prognostic significance as 2 cases of cerebral malaria had high OCT levels of 11.1 UAL and 10.7 IU/L, respectively.Key Words: Malaria, Ornithine carbamoyl transferase     

Characteristics of volunteers responding to emergencies in the Public Access Defibrillation Trial

OBJECTIVES: To evaluate the characteristics of volunteers responding to emergencies in the North American Public Access Defibrillation (PAD) Trial. METHODS: The PAD Trial was a prospective evaluation of cardiac arrest survival in community facilities randomized to cardiopulmonary resuscitation (CPR) or to CPR with automated external defibrillators (AEDs). The PAD volunteers' characteristics were analyzed using Poisson regression clustered on the facility and offset by the number of emergency episodes to which volunteers were exposed. RESULTS: A total of 19,320 volunteers in 1260 facilities were trained to provide emergency care. Of these, 8169 volunteers were participating actively at their facility during a time when one or more emergency episodes occurred. There were 1971 emergency episodes responded to by 1245 volunteers. The treatment arm (CPR-only versus CPR+AED) was not associated with the likelihood of volunteer participation in an episode. Likewise, the volunteers' age or sex did not affect response. Volunteers more likely to respond were supervisory/management or security personnel, non-minority participants, volunteers with previous CPR training, volunteers with previous experience in emergency care and those who passed the PAD CPR skills follow-up test. Volunteers who had a formal education beyond a high school level were less likely to respond. CONCLUSIONS: Volunteers with previous emergency training and positions of responsibility in their facility had a greater likelihood of participation in medical emergencies in the PAD Trial.     

Using the Exception from Informed Consent Regulations in Research

This article reflects the proceedings of a breakout session, “Using the Regulations in Research” at the 2005 Academic Emergency Medicine Consensus Conference, “Ethical Conduct of Resuscitation Research.” There have been two organized studies, and a number of anecdotal reports, describing the decline in cardiac arrest resuscitation research in the United States since the implementation of the Final Rule. Paradis and colleagues found that the volume of human cardiac arrest research published in the United States was significantly less in a four‐year period after the Final Rule was adopted as compared to the earlier period. Nichol and colleagues reported that both the absolute number of US‐based randomized cardiac arrest trials and the proportion of US‐based trials (vs. foreign trials, based on the mailing address of the first author) decreased by about 15% annually. Despite the concern about a negative impact, there are at least five published trials, one in progress and one in planning that have been or are being conducted under the regulations. Those completed include the Diaspirin Cross‐Linked Hemoglobin, Public Access Defibrillation, Multicenter Vest CPR, Brain‐CPR, and Pre‐Hospital Treatment of Status Epilepticus trials. Reports of how investigators met the regulations and their experience in doing so are reviewed. A summary table of the federal regulations is provided. Participants discussed what additional information and research about using the regulations would be helpful for the promotion of quality resuscitation and emergency care research in the United States. Areas suggested for further investigation include: impact on the quality as well as quantity of such research; current level of understanding of the regulations by investigators, regulatory/IRB personnel and potential subjects (the general public); costs incurred: additional time required for preparation, approval and conducting community consultation and public disclosure; impact on research on non–life‐threatening conditions; value and cost of a registry; use of a standard reporting template for issues regarding meeting the requirements in individual clinical trials; whether more specific guidance would be helpful or restrictive; what constitutes effective community consultation and public disclosure; and whether titration of community consultation and public disclosure based on the risk of the proposed intervention to subjects is feasible and acceptable.