Oxidative Stress in Zebrafish (Danio rerio) Sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line

Quality control of histamine and methacholine in diagnostic solutions with capillary electrophoresis

Solutions of histamine and methacholine bromide in different matrices for diagnostic purposes were analyzed for stability and quality control using capillary electrophoresis. Histamine (2-[4-imidazolyl]ethylamine) [CAS No. 51-45-6] was determined using a 0.1 M Tris-borate buffer of pH 8.3 with 5·10-5 M cetyltrimethylammonium bromide (CTAB) and 0.005% poly(vinyl alcohol) (PVA) and detected at 214 nm using clenbuterol [4-amino-a-(tert.-butylaminomethyl)-3,5-dichlorobenzyl alcohol] [37148-27-9] as an internal standard. Metacholine bromide (acetyl-ß-methacholine bromide) [333-31-3] was determined with a 0.01 M creatinine-chloride buffer of pH 4.85 and detected with indirect UV at 230 nm using potassium as an internal standard. Histamine solutions were stable for a prolonged period of time, whereas under enforced degradation conditions methacholine was hydrolyzed, yielding acetic acid and (tentatively) ß-methylcholine as reaction products

Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis

Capillary electrophoresis has become a powerful analytical tool for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. The use of replaceable polymer solutions increases the lifetime of the capillary, improves the repeatability of the migration times and enables pressure injection. When pressure injection is used, rather than electrokinetic injection, it is assured that a representative part of the sample is introduced into the capillary. The possible lower resolution, which is a side effect of the pressure injection, can be made up for by an increase of selectivity. Using fluorescent labels, it is possible to detect DNA with a fluorescence detector under both native conditions in double-stranded (ds) form, and under denaturing conditions in single-stranded (ss) form. When DNA is separated in its ss form, as is necessary for DNA sequencing, we observe an increased selectivity compared to separation of that sample in its ds form. In this work, we exploited this increased selectivity for the analysis of denatured PCR products. It was found that DNA separated in the ss form yields superior separation; that is, a given analysis can be achieved with the same resolution in a shorter separation time compared to dsDNA. Therefore, it is advisable to separate DNA in the ss form if high resolution, size dependent separation is required. The enhanced resolution achieved with DNA migrating in the ss form enabled the separation of allelic ladders of short tandem repeats with a difference of 4 base pairs in the 200 base pair range, with separation times no longer than 6 min

Peptide-Based Fluorescence Resonance Energy Transfer Protease Substrates for the Detection and Diagnosis of Bacillus Species

We describe the development of a highly specific enzyme-based fluorescence resonance energy transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp., among which are the members of the B. cereus group. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species. The rational design of the substrates was based on the fact that the presence of D-amino acids in the target is highly specific for bacterial proteases. The designed D-amino acids containing substrates appeared to be specific for B. anthracis but also for several other Bacillus spp. and for both vegetative cells and spores. With the use of mass spectrometry (MS), cleavage products of the substrates could be detected in sera of B. anthracis infected mice but not in healthy mice. Due to the presence of mirrored amino acids present in the substrate, the substrates showed high species specificity, and enzyme isolation and purification was redundant. The substrate wherein the D-amino acid was replaced by its L-isomer showed a loss of specificity. In conclusion, with the use of these substrates a rapid tool for detection of B. anthracis spores and diagnosis of anthrax infection is at hand. We are the first who present fluorogenic substrates for detection of bacterial proteolytic enzymes that can be directly applied in situ by the use of D-oriented amino acids

The effects of spinal cord stimulation on quality of life in patients with therapeutically chronic refractory angina pectoris

Objective. For patients with refractory angina pectoris, spinal cord stimulation (SCS) is a beneficial and safe adjuvant therapy. However, it has not yet been established whether SCS alters the quality of life (QoL) in these patients. Methods. In this study, 26 consecutive patients (age 61.3 +/- 7.0 years, 13 females, angina duration 12.7 +/- 6.0 years) were recruited. Social, mental, and physical aspects of QoL were determined by Nottingham Health Profile (NHP I), depression scale (CES-D), scoring of angina pectoris attacks and short-acting nitroglycerine intake, pain score on the Visual Analog Scale (VAS), perceived health percentage, Satisfaction With Life scale (SWLS), and one-aspect Linear Analog Self Assessment scale (LASA). QoL outcomes at baseline were compared with reference values from healthy subjects. Within-group changes and magnitude of changes (effect size, ES) were assessed after 3 months and 1 year of SCS. Results. Compared to healthy subjects, the patients had significantly worse scores at baseline on NHP, SWLS, and LASA. After 3 months of SCS, NHP I aspect pain (ES = 1.39), AP-score (ES = 0.85), perceived health percentage (ES = -0.80), NTG-use (ES = 1.08) and VAS-score (ES = 1.13) were all significantly improved (p 0.80). Conclusion. QoL in patients with refractory angina pectoris is poor. Both pain and health aspects of QoL improved significantly after 3 months of SCS. Social, mental, and physical aspects of QoL were found improved after 1 year of SCS