Reliability of Intracellular Water and Ion Distributions as Measured by X-Ray Microanalysis - A Review

X-ray microanalysis can be an important tool to reveal the spatial relationships between polyelectrolytes, ions, and water as they occur within cells and tissues in vivo. To reach this goal, at least two of these three closely interrelated variables should be measured independently. Moreover, the absence of systematic errors should be proven. The present review discusses the probability of artificial ion and water shifts between intracellular compartments due to the growth of dendritic ice crystals much larger than the cross-sectioned remnants commonly seen in frozen-dried sections. Considering the possible mechanism of ice crystal growth it is concluded that ions and water are not translocated over large distances. Moreover, problems associated with the preparation of a sample for water content estimations are discussed here. The importance of an appropriate pre-freezing treatment is highlighted, as is the importance of fast freezing. The risk of artificial water shifts between compartments with different freezing properties is discussed and the absence of clefts between compartments or haloes around them as seen in frozen-dried sections is taken as an appropriate criterion. Constancy of section thickness and retention of full hydration of cryosections are necessary prerequisites for many of the techniques and conditions to fulfill these requirements are given

Intracellular Water Distribution and Aging as Examined by X-Ray Microanalysis

The results reviewed here demonstrate that 1. the distribution of dry mass as observed in frozen-dried cryosections might be used as an unbiased measure of intracellular dry mass resp. water distributions in the tissue in vivo and 2. the well-known loss of water from cells during aging is solely due to a water loss from mitochondria without changes in the water content of all other components of the cell in the case of rat liver and heart muscle. The reason for the water loss might be increased counter ion binding by membrane-bound enzymes due to decreased fluidity of the inner mitochondrial membrane with aging rather than changes of the permeability of the membrane or chemical modifications of mitochondrial proteins or DNA. It is assumed that the observed changes lead to decreased intramitochondrial diffusion of substrates and to conformational changes of enzymes. This would decrease both the velocity and the binding constants of certain energy-supplying reactions and could therefore play an important role in the aging process

Changes of Ion and Water Content of Mouse Intestinal Cells After Pilocarpine and Isoproterenol Stimulation

Cytoplasmic monovalent ion and water contents in morphologically defined mice jejuna) cells were measured by X-ray microanalysis in order to gain insight into the cell-type specificity of intestinal electrolyte transport mechanisms. Ion and water contents were measured independently. It was found that in some cases net fluxes of ions and water do not correspond to the assumption of constant osmotic activity of cytoplasmic Na and K ions. Stimulation of secretion with the cholinergic secretagogue pilocarpine resulted in efflux of Cl- from and influx of K+ into crypt enterocytes containing small secretion granula (crypt A cells). No significant changes in ion concentrations were found in crypt enterocytes without secretion granula (crypt B cells). Crypt A cells were more likely to be stimulated by pilocarpine than crypt B cells, and the basolateral K+ efflux pathway in crypt A cells appeared to be rate-limiting. In villus enterocytes, pilocarpine stimulated Cl- efflux. Isoproterenol caused marked changes in the cytoplasmic Cl content of all epithelial cells. These changes were reversed by inhibition of adenylate cyclase by alloxan, with the sole exception of Cl increase in villus absorptive cells. The results are consistent with an cAMP-mediated stimulated secretion in crypt epithelial cells and a predominantly cAMP-independent stimulation of absorption in villus cells. The results obtained suggest a transcellular route of Cl absorption in the mouse jejunum

Element Concentrations in the Intestinal Mucosa of the Mouse as Measured by X-Ray Microanalysis

Subcellular ion distribution in villus, crypt, Paneth and smooth muscle cells of the mouse small intestine under resting conditions was investigated by X-ray microanalysis of ultrathin cryosections. In addition, the mass distribution was estimated by measuring the optical transmission of the compartments in transmission electron micrographs. Each cell type is characterized by a special composition in terms of the major monovalent ions Na, K, and Cl. In particular, among crypt epithelial cells, those cells containing small secretion granula (termed crypt A cells) also display cytoplasmic ion concentrations significantly different from crypt epithelial cells lacking secretion granula (crypt B cells). Monovalent ion concentrations in the cytoplasm of Paneth cells, muscle cells, and crypt epithelial cells lacking secretion granula are higher than expected from osmotic considerations. Hence, significant binding of ions to cytoplasmic polyelectrolytes is assumed in these cells. There are gradients of dry mass and K concentration from the luminal to the basal side of the cell, both in crypt and in villus cells. The terminal web in these cells is rich in Na and Cl. The elemental composition of the large, dark secretion granula in Paneth cells is similar to that of the small dark granula in crypt cells. However, the two morphologically different types of granula within the Paneth cells have a significantly different elemental composition, which might reflect maturation of secretion granula

Binding of Ions to Nuclear Chromatin

Ion concentrations in isolated lymphocyte nuclei subjected to KCl or MgCl2 media of varying ionic strength were measured by X-ray microanalysis. Values were corrected for the contribution of free ions by estimating the volume fraction of the water space morphometrically. The amount of bound cations and Cl was constant and independent of the widely varying free ion concentration. It is concluded that the mechanism of binding is counterion condensation but with limited cooperativity. In contrast to classical counterion condensation theory, the binding of ions occurs at oppositely charged clusters at the surface of the chromatin. Therefore, both cations and anions are bound and binding cannot be completely delocalized. The bound ions stabilize the basic chromatin fibre but are not involved in the regulation of the transition between the condensed and decondensed state. Using earlier data, we estimated the concentration of free cations in rat liver nuclei under in-vivo conditions to be in the order of about 80 mM

DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. It is triggered as a stereotypic response to unrepaired nuclear DNA damage or to uncapped telomeres. In addition to a direct role of nuclear DNA double-strand breaks as inducer of a DNA damage response, two more subtle types of DNA damage induced by physiological levels of reactive oxygen species (ROS) can have a significant impact on cellular senescence: Firstly, it has been established that telomere shortening, which is the major contributor to telomere uncapping, is stress dependent and largely caused by a telomere-specific DNA single-strand break repair inefficiency. Secondly, mitochondrial DNA (mtDNA) damage is closely interrelated with mitochondrial ROS production, and this might also play a causal role for cellular senescence. Improvement of mitochondrial function results in less telomeric damage and slower telomere shortening, while telomere-dependent growth arrest is associated with increased mitochondrial dysfunction. Moreover, telomerase, the enzyme complex that is known to re-elongate shortened telomeres, also appears to have functions independent of telomeres that protect against oxidative stress. Together, these data suggest a self-amplifying cycle between mitochondrial and telomeric DNA damage during cellular senescence

The Relationship between the Aging- and Photo-Dependent T414G Mitochondrial DNA Mutation with Cellular Senescence and Reactive Oxygen Species Production in Cultured Skin Fibroblasts

Mutations in the mitochondrial genome (mtDNA) are thought to be one of the causes of age-dependent cellular decline through their detrimental effects on respiration or reactive oxygen species (ROS) production. However, for many mutations, this link has not been clearly established. This study aimed to further investigate the phenotypic importance of a T414G mutation within the control region of mtDNA, previously shown to accumulate in both chronologically and photoaged human skin. We demonstrate that during dermal skin fibroblast replication in vitro in five separate cultures obtained from elderly individuals, the T414G mutant load can either increase or decrease during progressive cell division, implying the absence of consistent selection against the mutation in this context. In support of this, by utilizing a cell-sorting approach, we demonstrate that the level of the T414G mutation does not directly correlate with increased or decreased mtDNA copy number, or markers of cellular ageing including lipofuscin accumulation or ROS production. By consequence, the mutation can be distributed with a bias towards either the proliferating or senescent cell populations depending on the cell line. In conclusion, we propose that this particular mutation may have little effect on ROS production and the onset of cellular senescence in cultured fibroblasts

Tissue engineering strategies to bioengineer the ageing skin phenotype in vitro

Human skin ageing is a complex and heterogeneous process, which is influenced by genetically determined intrinsic factors and accelerated by cumulative exposure to extrinsic stressors. In the current world ageing demographic, there is a requirement for a bioengineered ageing skin model, to further the understanding of the intricate molecular mechanisms of skin ageing, and provide a distinct and biologically relevant platform for testing actives and formulations. There have been many recent advances in the development of skin models that recapitulate aspects of the ageing phenotype in vitro. This review encompasses the features of skin ageing, the molecular mechanisms that drive the ageing phenotype, and tissue engineering strategies that have been utilised to bioengineer ageing skin in vitro

Mitochondria in Cell Senescence:Is Mitophagy the Weakest Link?

Cell senescence is increasingly recognized as a major contributor to the loss of health and fitness associated with aging. Senescent cells accumulate dysfunctional mitochondria; oxidative phosphorylation efficiency is decreased and reactive oxygen species production is increased. In this review we will discuss how the turnover of mitochondria (a term referred to as mitophagy) is perturbed in senescence contributing to mitochondrial accumulation and Senescence-Associated Mitochondrial Dysfunction (SAMD). We will further explore the subsequent cellular consequences; in particular SAMD appears to be necessary for at least part of the specific Senescence-Associated Secretory Phenotype (SASP) and may be responsible for tissue-level metabolic dysfunction that is associated with aging and obesity. Understanding the complex interplay between these major senescence-associated phenotypes will help to select and improve interventions that prolong healthy life in humans. Search strategy and selection criteria: Data for this review were identified by searches of MEDLINE, PubMed, and references from relevant articles using the search terms “mitochondria AND senescence”, “(autophagy OR mitophagy) AND senescence”, “mitophagy AND aging” and related terms. Additionally, searches were performed based on investigator names. Abstracts and reports from meetings were excluded. Articles published in English between 1995 and 2017 were included. Articles were selected according to their relevance to the topic as perceived by the authors