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.     

Activation of mechanosensitive ion channels in plasma membrane of K562 cells

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.     

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.      

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.     

Inhibiting and stimulating effect of amiloride on potential-dependent cation channels in K562 cells

Non-voltage-gated ion channels play an essential role in cellular signalling and ionic homeostasis in nonexcitable cells. The patch clamp method in cell-attached configuration was used to search for the effects of amiloride and gadolinium (Gd3+) exerted on two types of voltage-insensitive cationic channels in plasma membrane of human leukemia K562 cells: Na-selective channels activated by actin disassembly, and mechanosensitive channels. Here we demonstrate that amiloride in high concentrations (1 mM) caused a full inhibition of mechanosensitive channels in K562 cells similarly to Gd3+ effect in micromolecular concentration range. Na-selective channels controlled by actin dynamics were shown to be unaffected by Gd3+ similarly as by amiloride. We also found that application of amiloride to the extracellular surface of membrane patch resulted in a significant increase in the activity of sodium channels. This unexpected stimulatory effect of amiloride may represent an unknown mechanism of activation of non-voltage-gated sodium channels. The data show an essential difference of the activation and blockage of these types of cation-selective channels.     

Flora Europaea Notulae Systematicae ad Floram Europaeam spectantes No. 20 Corrigenda et Addenda

Following the publication of the last of the series of Flora Europaea Notulae, No. 20 in the Botanical Journal of the Linnean Society , 76: 297–384 (1978), a number of additions or alterations have been drawn to our attention. These are published in continuation.     

Mechanism of epithelial sodium channel (ENaC) regulation by cortactin: involvement of dynamin

We have recently shown that epithelial sodium channels (ENaC) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. However, it has been also demonstrated that GTPase, dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. F. i., treatment of mpkCCD(c14) cells monolayers with dynasore (in concentrations of 10 and 100 microM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, the same concentrations of dynasore caused reduced currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel's activity and is not suitable for studies of a role of dynamin in the clathrin-mediated endocytosis of ENaC. This effect is most likely caused either by dynasore's toxic effect upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments designed to study the role of dynamin different plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin K44A transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC subunits only (control); additional transfection of cortactin in this system resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease if ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but apart from that dynamin and related processes also might be involved into ENaC regulation by cortactin.     

Use of inert gas jets to measure the forces required for mechanical gene transfection

Background

Abnormal blood glucose (BG) concentrations have been associated with increased morbidity and mortality in both critically ill adults and infants. Furthermore, hypoglycaemia and glycaemic variability have both been independently linked to mortality in these patients. Continuous Glucose Monitoring (CGM) devices have the potential to improve detection and diagnosis of these glycaemic abnormalities. However, sensor noise is a trade-off of the high measurement rate and must be managed effectively if CGMs are going to be used to monitor, diagnose and potentially help treat glycaemic abnormalities.

Aim

To develop a tool that will aid clinicians in identifying unusual CGM behaviour and highlight CGM data that potentially need to be interpreted with care.

Methods

CGM data and BG measurements from 50 infants at risk of hypoglycaemia were used. Unusual CGM measurements were classified using a stochastic model based on the kernel density method and historical CGM measurements from the cohort. CGM traces were colour coded with very unusual measurements coloured red, highlighting areas to be interpreted with care. A 5-fold validation of the model was Monte Carlo simulated 25 times to ensure an adequate model fit.

Results

The stochastic model was generated using ~67,000 CGM measurements, spread across the glycaemic range ~2-10?mmol/L. A 5-fold validation showed a good model fit: the model 80% confidence interval (CI) captured 83% of clinical CGM data, the model 90% CI captured 91% of clinical CGM data, and the model 99% CI captured 99% of clinical CGM data. Three patient examples show the stochastic classification method in use with 1) A stable, low variability patient which shows no unusual CGM measurements, 2) A patient with a very sudden, short hypoglycaemic event (classified as unusual), and, 3) A patient with very high, potentially un-physiological, glycaemic variability after day 3 of monitoring (classified as very unusual).

Conclusions

This study has produced a stochastic model and classification method capable of highlighting unusual CGM behaviour. This method has the potential to classify important glycaemic events (e.g. hypoglycaemia) as true clinical events or sensor noise, and to help identify possible sensor degradation. Colour coded CGM traces convey the information quickly and efficiently, while remaining computationally light enough to be used retrospectively or in real-time.