Image analysis of comet assay measurements

In the last decade the 'comet assay' or 'single cell gel electrophoresis assay' has been established as a sensitive method for the detection of DNA damage and the measurement of its recovery. The results published in the literature have often been obtained with different methods for comet structure measurement. In most cases these data are not comparable with each other. Even when using similar systems for the analysis, it is difficult to obtain matching data. This presentation will describe some technical aspects of our measurement equipment and evaluation software. It focuses on necessary experimental conditions to minimize errors in obtaining such data. The software developed here allows the rapid analysis of the microscopic samples (< 2 s per image). The image analysis was designed with respect to the morphological shapes of comet cells, which were investigated with a confocal laser microscope. The system is built with standard components which are commercially available. As a measure of the amount of DNA damage the ratio of fluorescence intensity was used inside the comet tail and the fluorescence intensity of the comet head. Other parameters such as DNA content, comet area, head radius, tail length and tail moment are also determined. The reproducibility of the system has been evaluated in several experiments over a period of 5 years.     

T cells of the human intestinal lamina propria are high producers of interleukin-10

BACKGROUND: The transmission of hepatitis A virus (HAV) has been associated with the use of a number of solvent/detergent-treated factor VIII concentrates and possibly a factor IX concentrate. These reports have emphasized the necessity of using virus-inactivation methods for plasma products that are capable of inactivating nonenveloped viruses such as HAV. STUDY DESIGN AND METHODS: A simple, highly accurate titration procedure for HAV, which allows extensive kinetic investigations of virus-inactivation procedures, has been developed. This system has now been used to evaluate the efficacy of vapor heating in inactivating HAV after the addition of the virus to a range of human plasma products. RESULTS: It was demonstrated that HAV was significantly more thermostable than other picornaviruses, which reinforced the fact that such viruses cannot be used as model viruses for HAV-inactivation studies. A one-step vapor-heating procedure was demonstrated to inactivate between 5.9 and > 6.3 log10 of HAV in different products. A two-step vapor-heating procedure had the capacity to inactivate between > 8.7 and > 10.4 log10 of HAV. Both procedures were more effective in inactivating HAV than was the pasteurization procedure used for virus inactivation in human albumin solutions. CONCLUSION: These data demonstrate the efficacy of vapor heating in inactivating high-titer HAV after the spiking of plasma products with virus. This study confirms and explains the results of controlled clinical trials and long-term clinical usage with respect to the lack of HAV transmission by such vapor-heated products.     

Actin and fimbrin are required for the internalization step of endocytosis in yeast

In Saccharomyces cerevisiae, alpha-factor is internalized by receptor-mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta-tubulin and actin mutant strains, we showed that actin plays a direct role in receptor-mediated internalization of alpha-factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta-tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin-binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha-factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.     

Crystallographic shear planes in nanocrystalline SnO2 thin films by high-resolution transmission electron microscopy

Atomic structures of crystallographic shear planes (CSPs) in nanocrystalline thin films of semiconductor SnO₂ were investigated by high-resolution electron microscopy. The films were prepared by electron beam evaporation in high vacuum (10^–6 torr) and followed by annealing in synthetic air at 700 °C for 1–2 H. CSPs with the displacement vector of [1/2 0 1/2] were observed in the planes parallel to (¯101), (110) and (¯3¯21). Most of the CPSs were found to terminate or interact with each other within SnO₂ crystallites. Partial dislocations exist at terminal places of CSPs or along intersecting lines of CSPs. CSP steps were also observed. Structural models of these defects have been proposed. Based on analysis of experimental data, it has been suggested that the Sn/O ratio at CSPs which are not parallel to their displacement vector, at cores of partial dislocations and at CSP steps, is higher than that of the perfect structure, that is, these defects are able to provide extra free electrons with the films.     

UNSTEADY-STATE SORPTION MEASUREMENTS A TEST OF DIFFERENT METHODS TO EVALUATE THE DIFFUSIVITY FROM UNSTEADY-STATE SORPTION MEASUREMENTS

Different simple methods for evaluating the diffusivity from a sorption measurement are presented and tested when there is a surface resistance (a limited mass transfer coefficient), a non-perfect step in relative humidity or non-Fickian effects. It is concluded that the different methods have different qualities and it is impossible to give a general recommendation on which method to use. The best tactic is probably to use more than one of the methods. A knowledge of the experimental conditions will help in choosing the best method.     

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.     

Embryotoxic doses of vitamin A to rabbits result in low plasma but high embryonic concentrations of all-trans-retinoic acid: risk of vitamin A exposure in humans

Retinoid pharmacokinetics were examined in plasma, placenta and embryos of gestational d 12 rabbits following application of an embryotoxic dosing regimen (10 mg retinyl palmitate/kg body wt per day from gestational d 7 to 12). Vehicle-treated or untreated rabbits served as controls. Physiological concentrations of all-trans-retinoic acid (all-trans-RA) and 13-cis-RA in rabbit plasma (5-8.33 nmol/L) were very close to the endogenous levels in human plasma. In addition, we identified endogenous all-trans-RA, 3,4-didehydroretinol and 3,4-didehydroretinoic acid in rabbit embryo. Following the last retinyl palmitate administration, apparent steady-state concentrations of all retinoids were reached in the examined compartments of rabbits. The major polar retinoid in plasma was 9, 13-di-cis-RA, but its embryonic concentrations were only about 6% of those in plasma. In the embryo, retinol and its esters were found at high concentrations; lower amounts of all-trans-4-oxo-RA and the newly identified 14-hydroxy-4, 14-retro-retinol could also be measured. Embryonic concentrations of all-trans-RA were about 100% higher than endogenous levels. The overall exposure of the embryo to this retinoid was, however, substantial. Embryonic area under the concentration time curve values strongly suggest that the embryotoxicity of the applied dosing regimen is mainly due to the action of all-trans-RA. A very remarkable finding of this study is the marginal increase of plasma concentrations of all-trans-RA over their endogenous levels, which is comparable to the human situation after vitamin A intake. This analogy indicates that high vitamin A intake may be associated with a higher risk for teratogenic effects in humans even in the absence of high elevation of plasma all-trans-RA levels.     

Topical use of aprotinin in open heart operations

Among various pharmacological agents used to reduce bleeding after open-heart operations, high-dose aprotinin therapy seems most promising. However, its long-term effects are still obscure; there is almost always possibility of bypass graft occlusions produced by the hypercoagulable state induced by aprotinin in coronary bypass operations. Topical application of aprotinin into the pericardial cavity could prevent the adverse effects. Fifty patients were prospectively studied to evaluate the effects of topical aprotinin. One million KIU of aprotinin was poured into the pericardial cavity before closure of the sternotomy in group 1 (n = 25). Patients in group 2 (n = 25) served as controls. Total postoperative bleeding was significantly reduced in group 1 when compared with that of group 2 (722.7 +/- 230.8 versus 1,282.6 +/- 225.7 mL; p < 0.01). The use of banked donor blood products was significantly less in group 1 than in group 2 (0.33 +/- 0.67 versus 1.36 +/- 0.86 units; p < 0.01). These results show that topical use of aprotinin reduces post-operative blood loss and need for transfusion. It seems promising and warrants further studies to be done.