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

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

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

Effect of Long-Term Hormone Therapy on Telomere Length in Postmenopausal Women

Telomeres undergo attrition with each cell division, and telomere length is associated with age-related diseases and mortality in the elderly. Estrogen can influence the attrition of telomeres by diverse mechanisms. This is a retrospective case control study that investigated the influence of long-term hormone therapy (HT) on telomere length in postmenopausal women. We recruited 130 postmenopausal women from 55 to 69 years of age for this study, and divided them into two groups. The first group included 65 women who had been on estrogen and progesterone therapy for more than five years (HT group). The other group was composed of 65 women matched in age to the HT group who had never had HT (non-HT group). The relative ratios of telomere length of study subjects to a reference DNA from a healthy young female were measured using quantitative PCR. Plasma levels of lipid profiles, total antioxidant status (TAS), C-reactive proteins (CRP), fasting glucose levels, and estradiol levels were measured. Age at menopause, vitamin use, and exercise, alcohol, and cigarette smoking histories were also assessed in a questionnaire. Mean duration (± SD) of HT was 8.4 ± 2.3 years. Prevalence of vitamin use and regular exercise were higher in the HT group than in the non-HT group (p<0.01). Relative telomere length ratios in the HT group were significantly greater than those in the non-HT group (p<0.01). HT was significantly correlated with the relative telomere length ratio in multivariate analysis when potential confounding variables were controlled for (p<0.05). In conclusion, telomere lengths were longer in postmenopausal women who had a history of long-term HT than in postmenopausal women without HT. Long-term HT in postmenopausal women may alleviate telomere attrition

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

Effective Field Theories in R_xi gauges

In effective quantum field theories, higher dimensional operators can affect the canonical normalization of kinetic terms at tree level. These contributions for scalars and gauge bosons should be carefully included in the gauge fixing procedure, in order to end up with a convenient set of Feynman rules. We develop such a setup for the linear R_xi-gauges. It involves a suitable reduction of the operator basis, a generalized gauge fixing term, and a corresponding ghost sector. Our approach extends previous results for the dimension-six Standard Model Effective Field Theory to a generic class of effective theories with operators of arbitrary dimension.Comment: 16 pages, no figure

Dietary Restriction Ameliorates Age-Related Increase in DNA Damage, Senescence and Inflammation in Mouse Adipose Tissuey.

Ageing is associated with redistribution of fat around the body and saturation of visceral adipose depots. Likewise, the presence of excess fat in obesity or during ageing places extra stress on visceral depots, resulting in chronic inflammation and increased senescence. This process can contribute to the establishment of the metabolic syndrome and accelerated ageing. Dietary restriction (DR) is known to alleviate physiological signs of inflammation, ageing and senescence in various tissues including adipose tissue. OBJECTIVES:Our pilot study aimed to analyse senescence and inflammation parameters in mouse visceral fat tissue during ageing and by short term, late-onset dietary restriction as a nutritional intervention. Design, measurements: In this study we used visceral adipose tissue from mice between 5 and 30 months of age and analysed markers of senescence (adipocyte size, γH2A.X, p16, p21) and inflammation (e.g. IL-6, TNFα, IL-1β, macrophage infiltration) using immuno-staining, as well as qPCR for gene expression analysis. Fat tissues from 3 mice per group were analysed. RESULTS:We found that the amount of γH2A.X foci as well as the expression of senescence and inflammation markers increased during ageing but decreased with short term DR. In contrast, the increase in amounts of single or aggregated macrophages in fat depots occurred only at higher ages. Surprisingly, we also found that adipocyte size as well as some senescence parameters decreased at very high age (30 months). CONCLUSIONS:Our results demonstrate increased senescence and inflammation during ageing in mouse visceral fat while DR was able to ameliorate several of these parameters as well as increased adipocyte size at 17.5 months of age. This highlights the health benefits of a decreased nutritional intake over a relatively short period of time at middle age