Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme.
(9/2521)
Tissue distribution and levels of allopurinol oxidizing enzyme and xanthine oxidase with hypoxanthine as a substrate were compared with supernatant fractions from various tissues of mice and from liver of mice, rats, guinea pigs and rabbits. The allopurinol oxidizing enzyme activities in liver were quite different among the species and the sex difference of the enzyme activity only in mouse liver. In mice, the highest activity of allopurinol oxidizing enzyme was found in the liver with a trace value in lung, but the enzyme activity was not detected in brain, small intestine and kidney, while the highest activity of xanthine oxidase was detected in small intestine, lung, liver and kidney in that sequence. The allopurinol oxidizing enzyme activity in mouse liver supernatant fraction did not change after storage at -20 degrees C or dialysis against 0.1 M Tris-HCl containing 1.15% KCl, but the activity markedly decreased after dialysis against 0.1 M Tris-HCl. On the contrary, the xanthine oxidase was activated 2 to 3 times the usual activity after storage at -20 degrees C or dialysis of the enzyme preparation. These results indicated that allopurinol was hydroxylated to oxipurinol mainly by the enzyme which is not identical to xanthine oxidase in vivo. A possible role of aldehyde oxidase involved in the allopurinol oxidation in liver supernatant fraction was dicussed. (+info)
Combinations of intervention treatments resulting in 5-log10-unit reductions in numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 organisms in apple cider.
(10/2521)
The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log10 units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log10 units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35 degrees C and/or freeze-thawing (48 h at -20 degrees C; 4 h at 4 degrees C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log10-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35 degrees C treatments. In pH 3.7 cider the 5-log10-unit reduction followed freeze-thawing combined with either 6 h at 4 degrees C, 2 h at 25 degrees C, or 1 h at 35 degrees C or 6 h at 35 degrees C alone. A 5-log10-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35 degrees C plus freeze-thawing, SA plus 12 h at 25 degrees C plus freeze-thawing, SA plus 6 h at 35 degrees C, and SA plus 4 h at 35 degrees C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35 degrees C. Cider with no added organic acids treated with either 6 h at 35 degrees C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement. (+info)
Pressure-induced syneretic response in rhesus monkey lenses.
(11/2521)
PURPOSE: To investigate the effect of pressure on the freezable and nonfreezable water content of the lens. METHODS: Excised rhesus monkey lenses in tissue culture media were subjected to three different hydrostatic pressures (2 atm, 1 atm, and 0.03 atm) for 24 hours. Then while still under the experimental pressure, the vessels were cooled in dry ice-acetone until the lenses were frozen. While the lenses were kept frozen, nuclear and cortical samples were dissected, enclosed in a sample pan, and weighed. Differential scanning calorimetry (DSC) measurements were performed between -30 degrees C and 30 degrees C. Total water content of each lens sample was obtained by thermogravimetric analysis at 105 degrees C. The nonfreezable water content was obtained by subtracting the freezable water content calculated from the DSC data from the total water content. RESULTS: The total water content of the lenses did not change significantly as a function of pressure applied. This was true both for cortical and for nuclear sections. The freezable water content increased as the pressure decreased both in cortex and nucleus. Similarly, the freezable water/nonfreezable water ratio also decreased with increasing pressure. CONCLUSIONS: External hydrostatic pressure would generate an influx of water into the lens. To alleviate this diluting tendency and to prevent turbidity as a result of dilution, the lens must effect an osmotic pressure change equivalent to the applied pressure. Change in the osmotic pressure is caused by changing the activity of the water (i.e., converting free water to bound water). This is a reversible and energetically the least expensive response. The release of bound water from the hydration layers of macromolecules and its conversion to free water in condensed systems are known as syneresis. In the lens decreasing pressures induce syneresis as demonstrated by the increase in freezable water content and the freezable water/nonfreezable water ratio. Such a response may be operative also in accommodating lenses. (+info)
Effect of multiple freeze-thaw cycles on hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA technology.
(12/2521)
Quantification of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA often is performed in specimens that have been frozen and thawed more than once. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles. We therefore evaluated the effect of multiple FT cycles on HBV DNA and HCV RNA quantification by testing serum specimens subjected to one (baseline), two, four, and eight FT cycles with the appropriate Chiron Quantiplex assay. Linear regression analysis showed minor increases of 1.7% per FT cycle for both HBV DNA and HCV RNA. The rise in HCV RNA levels was more pronounced among low-concentration samples, since further analysis revealed an increase of 3.2% per FT cycle among samples with 0.2 to 3.86 Meq of HCV RNA per ml. Given that the coefficient of variation for the Quantiplex assays is generally 10 to 15%, the minor increases in HBV DNA and HCV RNA levels with progressive FT cycles for the specimens tested were recognized only because analysis of variance revealed a statistically significant trend (P < 0.05). Due to the minor statistical trend, the clinical impact for individual patient specimens is likely to be limited, but it may deserve further study. In conclusion, the concentration of HBV DNA and HCV RNA in serum specimens subjected to up to eight short-term FT cycles was stable. (+info)
The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif.
(13/2521)
The sfr mutations, which result in sensitivity to freezing after cold acclimation, define genes that are required for freezing tolerance. We tested plants homozygous for mutations sfr2 to sfr7 for cold-induced gene expression and found that sfr 6 plants were deficient in cold-inducible expression of the genes KIN1, COR15a, and LTI78, which all contain the C repeat/dehydration-responsive element (CRT/DRE) motif in their promoters. Similarly, sfr 6 plants failed to induce KIN1 normally in response to either osmotic stress or the application of abscisic acid. In contrast, cold-inducible expression of genes CBF1, CBF2, CBF3, and ATP5CS1, which lack the CRT/DRE motif, was not affected. The freezing-sensitive phenotype that defines sfr 6 also was found to be tightly linked to the gene expression phenotype. To determine whether the failure of cold induction of CRT/DRE-containing genes in sfr 6 was due to altered low-temperature calcium signaling, cold-induced cytosolic-free calcium ([Ca2+]cyt) elevations were investigated in the sfr 6 mutant, but these were found to be indistinguishable from those of the wild type. We discuss the possibilities that CRT/DRE binding proteins (such as CBF1) require activation to play a role in transcription and that the SFR6 protein is a vital component of their activation. (+info)
Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.
(14/2521)
The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C. (+info)
Congruent docking of dimeric kinesin and ncd into three-dimensional electron cryomicroscopy maps of microtubule-motor ADP complexes.
(15/2521)
We present a new map showing dimeric kinesin bound to microtubules in the presence of ADP that was obtained by electron cryomicroscopy and image reconstruction. The directly bound monomer (first head) shows a different conformation from one in the more tightly bound empty state. This change in the first head is amplified as a movement of the second (tethered) head, which tilts upward. The atomic coordinates of kinesin.ADP dock into our map so that the tethered head associates with the bound head as in the kinesin dimer structure seen by x-ray crystallography. The new docking orientation avoids problems associated with previous predictions; it puts residues implicated by proteolysis-protection and mutagenesis studies near the microtubule but does not lead to steric interference between the coiled-coil tail and the microtubule surface. The observed conformational changes in the tightly bound states would probably bring some important residues closer to tubulin. As expected from the homology with kinesin, the atomic coordinates of nonclaret disjunctional protein (ncd).ADP dock in the same orientation into the attached head in a map of microtubules decorated with dimeric ncd.ADP. Our results support the idea that the observed direct interaction between the two heads is important at some stages of the mechanism by which kinesin moves processively along microtubules. (+info)
Cold-induced freezing tolerance in Arabidopsis.
(16/2521)
Changes in the physiology of plant leaves are correlated with enhanced freezing tolerance and include accumulation of compatible solutes, changes in membrane composition and behavior, and altered gene expression. Some of these changes are required for enhanced freezing tolerance, whereas others are merely consequences of low temperature. In this study we demonstrated that a combination of cold and light is required for enhanced freezing tolerance in Arabidopsis leaves, and this combination is associated with the accumulation of soluble sugars and proline. Sugar accumulation was evident within 2 h after a shift to low temperature, which preceded measured changes in freezing tolerance. In contrast, significant freezing tolerance was attained before the accumulation of proline or major changes in the percentage of dry weight were detected. Many mRNAs also rapidly accumulated in response to low temperature. All of the cold-induced mRNAs that we examined accumulated at low temperature even in the absence of light, when there was no enhancement of freezing tolerance. Thus, the accumulation of these mRNAs is insufficient for cold-induced freezing tolerance. (+info)