Tallimustine lesions in cellular DNA are AT sequence-specific but not region-specific.

Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.     

Comparison of several types of enrichment for captive felids

Enrichment can increase the complexity of the captive environment and possibly enhance captive animals' well‐being by stimulating active behaviors and reducing stereotypical behaviors commonly seen in zoo felids. In this study, three different enrichment items were added to outdoor enclosures of felids at the Montgomery Zoo to test their effects on activity levels and stereotypic pacing. Bones, frozen fish, and spices (cinnamon, chili powder, and cumin) were presented over a 3‐month period to six species of felids: cheetah, cougar, jaguar, lion, ocelot, and tiger. Proportion of time spent engaging in active behaviors and stereotypic pacing were compared before, during, and after treatments. All treatments resulted in a significant increase in activity level from baseline (bones: +15.59%; frozen fish: +35.7%; spices: +12.38%). Effects of enrichment items on activity levels were not sustained 7 days after removal. Proportion of time spent pacing significantly decreased during presentation of spices (?21.25%) and frozen fish (?26.58%), but not with the addition of bones. However, only the effect of frozen fish on stereotypic behavior was sustained 7 days after removal of the enrichment item. In conclusion, bones, spices, and frozen fish are inexpensive and easy‐to‐administer enrichment items that may be used to increase active behaviors of captive felids. Zoo Biol 26:371–381, 2007. © 2007 Wiley‐Liss, Inc.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

Comparison of tissue-cultured bovine endothelial cells from aorta and saphenous vein

Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

DNA methylation-associated colonic mucosal immune and defense responses in treatment-na?ve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.     

Enzymatic activity of endogenous telomerase associated with intact nuclei from human leukemia CEM cells.

Telomerase, a telomere-specific DNA polymerase and novel target for chemotherapeutic intervention, is found in many types of cancers. Telomerase activity is typically assayed using an exogenous primer and cellular extracts as the source of enzyme. Since the nuclear organization might affect telomerase function, we developed a system in which telomerase in intact nuclei catalyzes primer extension. Telomerase activity in isotonically isolated nuclei from human CEM cells shows low processivity (addition of up to four TTAGGG repeats). In contrast, telomerase activity which leaks into a 500 g postnuclear supernatant and the activity in a CHAPS extract are highly processive. The nucleotide inhibitor, 7-deaza-dGTP, seems to be more inhibitory against the nuclei-associated enzyme compared to telomerase from cytoplasmic extracts. However, 7-deaza-dATP and ddGTP are less inhibitory against nuclei-associated telomerase. The results suggest that the association of telomerase with the nuclear chromatin affects telomerase activity. Examination of telomerase activity in a more natural nuclear environment may shed new light on the telomerase function and provide a useful system for the evaluation of new telomerase inhibitors.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.