Simultaneous quantitation of 7-methyl- and O6-methylguanine adducts in DNA by liquid chromatography-positive electrospray tandem mass spectrometry

A methodology has been developed and validated for the simultaneous quantitation of O6-methyl- and 7-methylguanine in DNA isolated from in vitro exposure to the model alkylating agents: N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS). After exposure, DNA was isolated and directly hydrolyzed under acid conditions to hydrolytes containing DNA bases (modified and unmodified). The hydrolytes were used for direct O6- and 7-methylguanine quantitation using a rapid and selective liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS). The lower limits of quantitation for O6-methyl- and 7-methylguanine were 75.8 and 151.5 fmol, respectively. Linearity of the calibration curve was greater than 0.999 from 75.8 to 151,600.0 fmol for O6-methylguanine and 0.999 from 151.5 to 303,200.0 fmol for 7-methylguanine. The intra-day assay precision relative standard deviation (R.S.D.) values for O6-methylguanine for quality control (QC) samples were < or =9.2% with accuracy values ranging from 90.8 to 118%, and for 7-methylguanine the R.S.D. values for QC samples were < or =11%, with accuracy values ranging from 92.9 to 119%. The inter-day assay precision (R.S.D.) values for O6-methylguanine QC samples were < or =7.9% with accuracy values ranging from 94.5 to 116%, and for 7-methylguanine QC samples were < or =7.1% with accuracy values ranging from 95.2 to 110.2%. This method was used for simultaneous determination of the levels of 7-methyl- and O6-methylguanine in DNA acidic hydrolytes present in a series of incubations from salmon testis DNA treated with either MNU or MMS.     

Carbohydrate composition of lipoprotein apoproteins isolated from rat plasma and from the livers of rats fed orotic acid

Dietary orotic acid has been shown to inhibit secretion of plasma lipoproteins from the liver and to cause the accumulation of lipoprotein apoproteins within the liver. Comparison of the carbohydrate composition of plasma apoproteins and corresponding apoproteins isolated from orotic acid induced fatty livers indicates that the latter are deficient in N-acetyl glucosamine, galactose, and N-acetyl neuraminic acid. These differences can be explained by a lack of exposure of liver apoproteins to glycosylating enzymes present in the Golgi apparatus.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.     

Real-time three-dimensional transthoracic echocardiography in daily practice: initial experience

Aim of the work

To evaluate the feasibility and possible additional value of transthoracic real-time three-dimensional echocardiography (RT3D-TTE) for the assessment of cardiac structures as compared to 2D-TTE.

Methods

320 patients (mean age 45 ± 8.4 years, 75% males) underwent 2D-TTE and RT3D-TTE using 3DQ-Q lab software for offline analysis. Volume quantification and functional assessment was performed in 90 patients for left ventricle and in 20 patients for right ventricle. Assessment of native (112 patients) and prosthetic (30 patients) valves morphology and functions was performed. RT3D-TTE was performed for evaluation of septal defects in 30 patients and intracardiac masses in 52 patients.

Results

RT3D-TTE assessment of left ventricle was feasible and reproducible in 86% of patients while for right ventricle, it was (55%). RT3D-TTE could define the surface anatomy of mitral valve optimally (100%), while for aortic and tricuspid was (88% and 81% respectively). Valve area could be planimetered in 100% for the mitral and in 80% for the aortic. RT3D-TTE provided a comprehensive anatomical and functional evaluation of prosthetic valves. RT3D-TTE enface visualization of septal defects allowed optimal assessment of shape, size, area and number of defects and evaluated the outcome post device closure. RT3D-TTE allowed looking inside the intracardiac masses through multiple sectioning, valuable anatomical delineation and volume calculation.

Conclusion

Our initial experience showed that the use of RT3D-TTE in the assessment of cardiac patients is feasible and allowed detailed anatomical and functional assessment of many cardiac disorders.     

Sprint interval training (SIT) is an effective method to maintain cardiorespiratory fitness (CRF) and glucose homeostasis in Scottish adolescents

The present study examined the physiological impact of a school based sprint interval training (SIT) intervention in replacement of standard physical education (SPE) class on cardio-respiratory fitness (CRF) and glucose homeostasis during the semester following summer vacation. Participants (n=49) were randomly allocated to either intervention (SIT; n=26, aged 16.9 ± 0.3 yrs) or control group who underwent standard physical education (SPE; n=23, aged 16.8 ± 0.6 yrs). CRF (VO₂max) and glucose homeostasis were obtained prior-to and following 7 weeks of SIT exercise. Significant group x time interaction was observed for CRF (P < 0.01) with non-significant trends for fasting insulin (P= 0.08), and HOMA-IR (P=0.06). CRF decreased (P < 0.01) in SPE such that POST intervention CRF was significantly lower (P< 0.05) in SPE. Fasting plasma glucose (P < 0.01), insulin (P< 0.01) and HOMA-IR (P< 0.01) increased significantly amongst SPE. The main finding of the present study is that 7-weeks of SIT exercise is an effective method of maintaining (but not improving) CRF and fasting insulin homeostasis amongst school-going adolescents. SIT exercise demonstrates potential as a time efficient physiological adjunct to standard PE class in order to maintain CRF during the school term.     

Durable remissions are rare following high dose therapy with autologous stem cell transplantation for adults with "paediatric" bone and soft tissue sarcomas

BACKGROUND: The role of high dose therapy (HDT) with autologous stem cell transplantation (AuSCT) for the treatment of bone and soft tissue sarcomas remains investigational. There are few reports examining this strategy focusing on the adult population. METHODS: We retrospectively reviewed our experience of adult patients undergoing HDT and AuSCT for 'paediatric' sarcomas. RESULTS: A total of 17 patients (14 male, 3 female) with median age at transplant of 24 years (range 20 - 41) were identified. The diagnosis was Ewings sarcoma/PNET (10), osteosarcoma (5) and rhabdomyosarcoma (2). Status prior to HDT, following conventional-dose chemotherapy +/- surgery +/- radiotherapy, was complete remission (CR) (6), partial remission (PR) (6), stable disease (1) and progressive disease (4). There was no transplant-related mortality. Two patients remain disease free beyond four years and both received HDT as part of their primary therapy (CR1 and PR1) however, the median progression free survival and overall survival following AuSCT for the entire cohort was only 7 months (range: 2-92 months) and 13 months (range: 2 - 92 months), respectively. CONCLUSION: HDT and AuSCT infrequently achieves prolonged remissions in adult patients and should only be considered in patients who are in a PR or CR following conventional-dose therapy. Further studies are required to define the role of HDT with AuSCT for adult patients with sarcoma.     

Apoptosis and p53 expression in rat adjuvant arthritis

The kinetics of apoptosis and the apoptosis-regulating gene p53 in adjuvant arthritis (AA) were investigated to assess the value of the AA rat model for testing apoptosis-inducing therapies. Very few terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were detected during the early phases of AA, but on day 23 (chronic arthritis) the percentage of TUNEL-positive cells was significantly increased. Expression of p53 in synovial tissue gradually increased from days 5-23, which was markedly higher than p53 levels in rheumatoid arthritis (RA) synovium. Significant apoptosis only occurs late in rat AA and is concordant with marked p53 overexpression, making it useful model for testing proapoptotic therapies, but rat AA is not the best model for p53 gene therapy because dramatic p53 overexpression occurs in the latter stages of the disease.     

Genome analysis of DNA repair genes in the alpha proteobacterium <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Background  

The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.