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.     

Production of recombinant endostatin in milk from transgenic mice

Using the bovine alpha S1-casein gene, a genetic construct with an endostatin-coding fragment of the mouse collagen XVIII cDNA was designed to express endostatin in milk of transgenic animals. Several transgenic mice were obtained. The mice secreted endostatin in milk at 70-300 ng/microliter and transmitted this character to their progeny.     

Expression analysis of proteins encoded by genes of the tag7/tagL (PGRP-S,L) family in human peripheral blood cells

Various types of human blood cells were tested for expression of the Tag7/PGRP-SA and TagL/PGRP-L proteins, which belong to the family of proteins possessing the lysozyme-like peptidoglycan recognition protein (PGRP) domain. Expression regulation by several factors was demonstrated.     

Production of Recombinant Endostatin in Milk of Transgenic Mice

Using the bovine S1-casein gene, a genetic construct with an endostatin-coding fragment of the mouse collagen XVIII cDNA was designed to express endostatin in milk of transgenic animals. Several transgenic mice were obtained. The mice secreted endostatin in milk at 70–300 ng/l and transmitted this character to their progeny.     

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.      

A simple and rapid method of determining the titer of herpes simplex virus antibodies in a study by immunoenzyme analysis of a single serum dilution

The result obtained in the study of the possibility of using the method for the determination of the titer of antibodies to herpes simplex virus by EIA techniques in a single dilution of the serum under test are presented. This method is based on the determination of the optical density of the serum titer (rcut) in different groups of sera with the use of the assay system, permitting the evaluation of the positive results obtained in the determination of their final dilution. The results obtained with the use of this method showed that error was 50% for high-titer sera, 60% for medium-titer sera and 30% for low-titer sera.     

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.     

Functional properties of the WNT11 new isoform, expressed in colon carcinoma cell line HT29

Colon carcinoma is a common type of neoplastic transformation. Mechanisms of its establishment and progression have been studying for several decades. Aberrant activation of the canonical Wnt signaling is frequently observed in colon carcinoma cells. Moreover, expression of the "noncanonical" Wnt ligands is also detected in this type of cancer. However, the implication of the noncanonical Wnt signaling in carcinogenesis and colorectal cancer (CRC) progression is still unclear. Here, to elucidate the characteristic features of the noncanonical Wnt signaling activation in CRC the expression of the "noncanonical" ligand hWnt11 has been studied. It was shown for the first time that expression of the hWnt11 in CRC is accompanied by the alternative splicing. The new hWnt11 isoform (hWnt11sp3) has been identified. Unlike to hWnt11, this isoform is not secreted and lacks the ability to inhibit the canonical Wnt signaling. Considering the canonical Wnt signaling inhibiting activity of hWnt11, different functional properties of the ligand and its isoform may reflect a special role of the alternative splicing in carcinogenesis and tumor progression. Thus, due to the difference in their functional properties an existence of several Wnt isoforms should be taken into account for the investigation of the role of Wnt ligands.