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.     

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.     

Sulfate reduction and microbial processes of the methane cycle in the sediments of the Sevastopol Bay

The rates of microbial processes of sulfate reduction and of the methane cycle were measured in the bottom sediments of the Sevastopol basin, where seeps of gaseous methane have been previously found. Typically for marine environments, sulfate reduction played the major role in the terminal phase of decomposition of organic matter (OM) in reduced sediments of this area. The rate of this process depended on the amount of available OM. The rate of methanogenesis in the sediments increased with depth, peaking in the subsurface horizons, where decreased sulfate concentration was detected in the pore water. The highest rates of sulfate-dependent anaerobic methane oxidation were found close to the methane-sulfate transition zone as is typical of most investigated marine sediments. The data on the carbon isotopic composition of gaseous methane from the seeps and dissolved CH₄ from the bottom sediments, as well as on the rates of microbial methanogenesis and methane oxidation indicate that the activity of the methane seeps results from accumulation of biogenic methane in the cavities of the underlying geological structures with subsequent periodic release of methane bubbles into the water column.     

The effect of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> red pigment on the expression of cloned human α-synuclein

Two transgenic yeast strains expressing human α-synuclein were used to study the impact of yeast red pigment exhibiting antiamyloid properties. It has been demonstrated that the endogenous red pigment produced under special conditions in strains carrying an ade1 mutation inhibits the expression of the hybrid protein α-synuclein-GFP. This was evident from the reduced mean value of GFP fluorescence and diminished number of cells accumulating cytoplasmic inclusions of α-synuclein-GFP. Exogenous forms of the purified red pigment (natural, synthetic and hydrolyzed derivatives) differ from the endogenous red pigment by their effect on α-synuclein. Exogenous red pigments increased the number of both cells expressing GFP fluorescence and those containing cytoplasmic inclusions. However, both endogenous and exogenous red pigments reduced the cloned α-synuclein toxicity and resulted in redistribution of the α-synuclein in cells. α-Synuclein content decreased in cell lysate pellets and increased in supernatants.     

Choline phospholipid metabolites of human vascular endothelial cells altered by cyclooxygenase inhibition,growth factor depletion,and paracrine factors secreted by cancer cells

Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO), reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs). We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.     

Estimating of changes in the amyloid and prion content of yeast cells

An attempt was made at estimating the overall amyloid content of yeast cells by treating crude cellular lysates with thioflavin T, the agent specifically staining amyloid fibrils. We demonstrated that overproduction of the yeast chaperone Hsp104p, as well as GuHCI treatment of the [PSI+] cells led both to elimination of the [PSI+] factor and to a stable decrease of the overall amyloid content estimated by intensity of fluorescence (IF) of the thioflavin T. At the same time, overexpression of gene SUP35, coding the protein prionizable to [PSI+], led to generation of [PSI+] clones with higher IF of thioflavin T. Cytoduction in the crosses involving PSI factor leads to considerable enhancement of IF; cytoductants with the nucleus of the recipient [psi-] strain not only got [PSI+] factor from the donor strain but also increased their amyloid content. In these model experiments all treatments modifying one of the yeast prions, [PSI+] factor, led to a predictable shift of IF of thioflavin T that behaved like a cytoplasmic hereditary determinant. The data obtained show that IF of thioflavin T staining gives reliable estimates of cellular amyloid content and that mitotically stable shift of IF after a battery of treatments modifying cellular prion set provides quantitative estimate of the input of prionizable protein molecules to the amyloid pool. The combination of thioflavin staining and prionotropic treatments applied here can be possibly used for future attempts of checking yeast strains for cryptic prions.     

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.      

Molecular genetic analysis of the X-chromosomal nuclear envelope attachment region in nurse cells of the malaria mosquitoes Anopheles messeae Fall

DNA of the X-chromosomal nuclear envelope attachment region was isolated from malaria mosquito Anopheles messeae Fall. nurse cells by chromosome microdissection. A DNA library of the region was constructed using a plasmid vector. DNA sequencing revealed gene fragments, tandem repeats, and a great variety of transposable elements (TEs). The X-chromosomal nuclear envelope attachment region was concluded to correspond in molecular organization and cytogenetics to diffuse intercalary heterochromatin.     

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.