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.     

Predictive Observation-Based Endpoint Criteria for Mice Receiving Total Body Irradiation

Total body irradiation of mice is a commonly used research technique; however, humane endpoints have not been clearly identified. This situation has led to the inconsistent use of various endpoints, including death. To address this issue, we refined a cageside observation-based scoring system specifically for mice receiving total body irradiated. Male and female C57BL/6 mice (age, 8 wk) received 1 of 3 doses of radiation from 1 of 2 different radiation sources and were observed for progression of clinical signs. All mice were scored individually by using cageside observations of their body posture (score, 0 to 3), eye appearance (0 to 3), and activity level (0 to 3). Retrospective analysis of the observation score data indicated that death could be predicted accurately with total scores of 7 or greater, and observation scores were consistent between observers. This scoring system can be used to increase the consistent use of endpoint criteria in total body murine irradiation studies and ultimately to improve animal welfare.Abbreviations: TBI, total body irradiation; ARS, acute radiation syndromeTotal body irradiation (TBI) of mice is a widely used technique with numerous applications. Mice undergoing TBI followed by stem cell transplant are a model of engraftment kinetics^13,14,17,18 and graft-versus-host disease.^10,28 Mice may undergo multiple rounds of TBI followed by gene transfer to create chimeras to study various diseases.^9,11,19,36 More basically, the biologic effects of radiation in mice are a useful model for the various manifestations of human radiation sickness^15,29,35,37 and can be used to develop and evaluate treatments.^5,23,37Despite the widespread irradiation of mice, studies typically do not report criteria used for humane euthanasia or the mortality observed due to irradiation with failure of the graft, transplant, or treatment. This practice has resulted in the frequent and widespread use of death or the moribund state as the experimental endpoint.^5,6,35 The moribund condition, an unresponsive and immobile animal, is a commonly used endpoint for a variety of research protocols associated with high mortality or progressive and severe disease states.^32,33 Using the moribund criterion requires the animal to progress through all potential phases of pain and distress associated with the chosen model to a near-death state before the animal is euthanized.²⁵The ability to predict death with a high probability and high accuracy for mice receiving TBI would allow preemptive euthanasia. This action would ameliorate terminal pain and distress associated with acute radiation syndrome (ARS) and markedly improve animal welfare yet maintain scientific integrity in accordance with the recommendation of the Guide for the Care and Use of Laboratory Animals.¹² Although objective endpoint criteria are generally preferable, this option is not always possible. For mice receiving TBI, neither weight loss²⁶ nor decreased food and water consumption²⁹ are good predictors of death, because many animals that survive demonstrate marked weight loss and reduced consumption. Furthermore, mice receiving TBI should be handled minimally to avoid increasing mortality.^15,31 As an alternative approach to objective endpoint criteria, subjective behavioral criteria may be used, providing that they are adequately described, monitored, and applied so that trends can be documented.^24,34The current study sought to refine an observation-based scoring system to assess the health status of irradiated mice and determine the broad applicability of endpoints across radiation doses, radiation sources, and animal sexes. It was performed in conjunction with an IACUC-approved study designed to establish survivability curves for mice undergoing TBI with no other experimental manipulation. Male and female C57BL/6 mice that received 1 of 3 TBI doses from 1 of 2 different radiation sources were evaluated by using daily cageside observational scoring of body posture, activity level, and eye appearance. The results were analyzed to verify consistency between observers and identify the predictive nature of these criteria of impending death, with the goal of establishing useful endpoint criteria for all mice receiving TBI.     

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.     

Crystal structure of LAAO from Calloselasma rhodostoma with an L-phenylalanine substrate: insights into structure and mechanism

L-Amino acid oxidase is a dimeric glycosylated flavoenzyme, a major constituent of the venom-from the snake Calloselasma rhodostoma. The enzyme exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities. The structure of l-amino acid oxidase with its substrate (L-phenylalanine) has been refined to a resolution of 1.8 A. The complex structure reveals the substrate bound to the reduced flavin (FADred). Alternative conformations for the key residues His223 and Arg322 are evident, suggesting a dynamic active site. Furthermore, conformational changes are apparent for the isoalloxazine ring; the three-ring system exhibits more bending around the N5-N10 axis compared to the oxidized flavin. The implications of the observed dynamics on the mechanism of catalysis are discussed. Inspection of buried surfaces in the enzyme reveals a Y-shaped channel system extending from the external surface of the protein to the active site. One portion of this channel may serve as the entry path for O2 during the oxidative half-reaction. The second region, separated from the proposed O2 channel by the N terminus (residues 8-16) of the protein, may play a role in H2O2 release. Interestingly, the latter portion of the channel would direct the H2O2 product to the exterior surface of the protein, near the glycan moiety, thought to anchor the enzyme to the host cell. This channel location may explain the ability of the enzyme to localize H2O2 to the targeted cell and thus induce the apoptotic effect.     

Growth,photosynthesis, and metabolism of sugar beet at an early stage of exposure to elevated CO<Subscript>2</Subscript>

The effects of CO₂ concentration (C _a) on growth, photosynthesis, and the activity of enzymes associated with the translocation and assimilation of CO₂ were studied in sugar beet (Beta vulgaris L. subsp. saccharifera, cv. Ramonskaya) plants. The plants were grown in controlled-climate chamber to the stage of 3–4 leaves and then used in experiments. Experimental plants were exposed in boxes to doubled C _a (700 µl/l, 2C plants), whereas control plants were kept in a chamber with ambient atmosphere (350 µl/l, 1C plants). As compared with 1C plants, in 3 and 8 days, the leaf area of 2C plants increased by 14 and 26%, respectively. The rate of their photosynthesis (P _n) measured in 3, 6, and 8 days increased by 85, 47, and 52%, respectively, whereas in normal air, the values of P _n in 2C plants were by 12, 19, and 15% lower than in 1C plants. After 8-day growth, the content of soluble carbohydrates in the leaves of 2C plants attained 7.2%, being by 80% greater than in 1C plants; the content of starch did not exceed 3%. The total content of chlorophylls a and b in the leaves of 2C plants was by 14% greater than in 1C plants, but their ratio was essentially the same. The level of protein in 2C plants was by 13.4% lower than in 1C plants. The activity and content of Rubisco in 1C and 2C plants were similar. As compared with 1C plants, in 2C plants the activity of soluble carbonic anhydrase (sCA) was lower by 34% in 3 days and by 18% in 8 days; the activity of carbonic anhydrase of membrane preparations (mCA), was lower by 24 and 77%, respectively. Catalase activity in 2C plants became by 8% lower than in 1C plants only after 8 days. A reduction in the photosynthetic ability of 2C plants in ambient atmosphere, a decrease in activity of sCA and, especially, of mCA observed together with invariable activity and content of Rubisco in the leaf extracts are interpreted as early symptoms of acclimation of young plants of sugar beet to elevated CO₂.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 184–190.Original Russian Text Copyright © 2005 by Ignatova, Novichkova, Mudrik, Lyubimov, Ivanov, Romanova.This revised version was published online in April 2005 with a corrected cover date.     

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.      

The Use of Phosphite‐Type Ligands in the Ir‐Catalyzed Asymmetric Hydrogenation of Heterocyclic Compounds

A series of chiral phosphite‐type ligands was tested in asymmetric Ir‐catalyzed hydrogenation of quinolines and 2,4,5,6‐tetrahydro‐1H‐pyrazino(3,2,1‐j,k)carbazole. Hydrogenation of quinaldine hydrochloride provided superior enantioselectivity up to 65% ee compared to quinaldine free base. The ligands were tested for the first time in the asymmetric Ir‐Ircatalyzed hydrogenation of 2,4,5,6‐tetrahydro‐1H‐pyrazino(3,2,1‐j,k)carbazole yielding the antidepressant drug, pirlindole. Chirality 26:56–60, 2013. © 2013 Wiley Periodicals, Inc.     

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 &lt; 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.