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Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21(WAF1/Cip) or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21(WAF1/Cip) was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-β expression at mRNA and protein levels also showed a rapid increase in TGF-β2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-β receptor. These data suggest that stress induced production of TGF-β2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.

In order to assess whether markers of cell senescence are related to reproductive failure, the expression of telomerase and telomere length in endometrial biopsies from women with and without reproductive failure were assessed. This pilot study included 45 women of whom 10 had idiopathic recurrent loss of empty gestational sacs, 10 had idiopathic recurrent fetal loss (miscarriage following identification of fetal cardiac activity), 10 had recurrent implantation failure and 15 had two or more normal pregnancies (control group). An endometrial sample was collected during the window of implantation from each woman. The mean endometrial telomere length was determined by quantitative polymerase chain reaction. Telomerase expression was evaluated by immunohistochemistry. The endometria of the control group showed virtually no telomerase immunoreactivity during the window of implantation. However, the immunostaining for telomerase was significantly and differentially increased in various endometrial cellular compartments in women with recurrent reproductive failure (P < 0.05). There were no significant differences in mean telomere length between groups. These data provide a novel insight into the biological correlates of clinical types of recurrent reproductive failure and suggest that specific alterations in the regulation of endometrial cell fate are associated with different types of recurrent reproductive failure.

Embryonic stem cells (ESCs) are capable of extended self-renewal and maintenance of pluripotency even after many population doublings. This is supported by high levels of telomerase activity and enhanced antioxidant protection in ESCs, both of which are downregulated during differentiation. To examine the role of telomerase for ESC self-renewal and differentiation, we overexpressed the reverse transcriptase subunit (Tert) of murine telomerase in ESCs. Increased telomerase activity enhances the self-renewal ability of the Tert-overexpressing ESCs, improves their resistance to apoptosis, and increases their proliferation. The differentiated progeny of wild-type ESCs express little Tert and show shortening of telomeric overhangs. In contrast, the progeny of Tert-overexpressing ESCs maintain high telomerase activity, as well as the length of G-rich overhangs. In addition, these cells accumulate lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Finally, differentiation toward hematopoietic lineages is more efficient as a result of the continued expression of Tert. Microarray analysis revealed that overexpression of Tert altered expression of a variety of genes required for extended self-renewal and lifespan. Our results suggest that telomerase functions as a “survival enzyme” in ESCs and its differentiated progeny by protecting the telomere cap and by influencing the expression patterns of stress response and defense genes. This results in improved proliferation of ESCs and more efficient differentiation, and these results might have profound consequences for stem cell–replacement therapies.

Summary Background : The chromosome instability observed in peripheral blood lymphocytes in ulcerative colitis could be a biomarker of cancer susceptibility. Aim : To determine whether accelerated telomere shortening could explain chromosome instability and assess the effect of drugs and smoking on telomere dynamics in these cells. Methods : Peripheral blood lymphocytes were isolated from ulcerative colitis, Crohn's disease and non-inflammatory bowel disease control patients. Telomere lengths were measured by quantitative real-time polymerase chain reaction. After activation and cell separation, telomerase activity and human telomerase reverse transcriptase messenger ribonucleic acid were measured by telomerase repeat amplification protocol enzyme-linked immunosorbent serological assay and quantitative real-time polymerase chain reaction, respectively. Results : Age-related telomere loss in peripheral blood lymphocytes was similar in ulcerative colitis, Crohn's disease and control patients. Telomerase activity decreased with age in all groups and correlated positively with telomere length (r = 0.489, P =0.006). Among Crohn's disease patients, azathioprine was associated with decreased telomerase activity (0.66 vs. 1.54, P = 0.026, P

Healthy ageing across the life course (HALCyon) is an interdisciplinary research collaboration that harnesses the power of nine UK cohort studies to discover life course influences on physical and cognitive capability, social and psychological well-being, and underlying biology. In this symposium, HALCyon co-investigators reported the first wave of findings from five of the eight work packages.

Objective To investigate the associations between low and high concentrations of baseline serum 25-hydroxyvitamin D [25(OH)D] and all-cause mortality in very old (≥85 years) men and women over 6 years. Design, setting and subjects Prospective mortality data from 775 participants in the Newcastle 85+ Study were analysed for survival in relation to 25(OH)D (season-specific quartiles and predefined cut-off values) and sex using Cox proportional hazards models. The models were fitted to the entire and restricted (nonusers of vitamin D-containing supplements and medication) cohorts. Results For the entire cohort, mortality was higher in both the lowest and highest 25(OH)D season-specific quartiles [SQ1: hazard ratio (HR) 1.31, 95% confidence interval (CI) 1.01–1.69, P = 0.04; SQ4: HR 1.44, 95% CI 1.12–1.85, P = 0.004] compared with the combined middle quartiles (SQ2 + SQ3), after adjustment for sociodemographic factors. The increased risk for the highest quartile remained significant after further adjustment for lifestyle variables (SQ4: HR 1.37, 95% CI 1.06–1.77, P = 0.02) and was seen only in women in sex-specific analyses. Similarly, in sensitivity analyses with predefined 25(OH)D cut-off values, the highest 25(OH)D concentration (≥75 nmol L−1) was associated with a 2.4-fold increased risk of mortality in women (restricted cohort) after adjusting for all covariates. Conclusion Low and high season-specific 25(OH)D quartiles were associated with increased risks of mortality over 6 years in the very old; this effect was particularly noticeable in women, including those who reported taking vitamin D-containing supplements/medication.

Telomeres protect the ends of all linear chromosomes against DNA loss and faulty recombination. They shorten during replication and also in response to external stress and damage. Telomerase counteracts telomere shortening but has nontelomeric functions as well. Shortened or otherwise uncapped telomeres are recognized by the cell's DNA damage response machinery and induce apoptosis or cell senescence, thus contributing to functional decline during aging. In humans and many other animals, telomeres in somatic tissues shorten with age. Telomere length in such tissues is associated with aging but currently lacks the diagnostic precision needed for it to be a reliable biomarker. Telomeres and telomerase, however, are promising targets for interventions to limit tumor growth and possibly to slow down age-related functional decline.

Replicative senescence of human diploid fibroblasts (HDFs) or melanocytes is caused by the exhaustion of their proliferative potential. Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Cells in replicative senescence share common features with cells in SIPS: morphology, senescence-associated beta-galactosidase activity, cell cycle regulation, gene expression and telomere shortening. Telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative damage. SIPS could be a mechanism of accumulation of senescent-like cells in vivo. Melanocytes exposed to sublethal doses of UVB undergo SIPS. Melanocytes from dark- and light- skinned populations display differences in their cell cycle regulation. Delayed SIPS occurs in melanocytes from light-skinned populations since a reduced association of p16(Ink-4a) with CDK4 and reduced phosphorylation of the retinoblastoma protein are observed. The role of reactive oxygen species in melanocyte SIPS is unclear. Both replicative senescence and SIPS are dependent on two major pathways. One is triggered by DNA damage, telomere damage and/or shortening and involves the activation of the p53 and p21(waf-1) proteins. The second pathway results in the accumulation of p16(Ink-4a) with the MAP kinase signalling pathway as possible intermediate. These data corroborate the thermodynamical theory of ageing, according to which the exposure of cells to sublethal stresses of various natures can trigger SIPS, with possible modulations of this process by bioenergetics.

Replicative senescence is characterized by the irreversible loss of division potential of cultivated human and animal cells. Correlations between the replicative potential in vitro and the age of the donor or the maximal lifespan of the species suggest replicative senescence to be an appropriate model for aging. Telomeres of human somatic cells shorten with each cell division but are stabilized at constant length in tumors and immortal cells by the enzyme telomerase. The assumption of a causal role of telomere shortening for the limited lifespan of cells in vitro was borne out recently. We could demonstrate oxidative stress as a main reason for telomere shortening. Telomeres are sensors for oxidative damage in the genome. Telomeres shorten during in vivo aging as well; however, there are significant differences between individuals. Telomere erosion might play a major role for the aging of the immune system. Our data suggest that telomere shortening in vivo could reflect the cumulative amount of oxidative damage to the organism. It might be useful as a biomarker of aging.

Study question Can the basal epithelial compartment of the human endometrium be defined by specific markers? Summary answer Human endometrial epithelial cells from the basalis express nuclear SOX9 and the cell-surface marker SSEA-1, with some cells expressing nuclear β-catenin. In vitro, primary endometrial epithelial cells enriched for SSEA-1+ show some features expected of the basalis epithelium. What is known already The endometrial glands of the functionalis regenerate from the basalis gland stumps following menstruation. Endometriosis is thought to originate from abnormal dislocation of the basalis endometrium. In the highly regenerative intestinal epithelium, SOX9 and nuclear β-catenin are more highly expressed in the intestinal crypt, the stem/progenitor cell region. Study design, size, duration A large prospective observational study analysing full-thickness human endometrial hysterectomy samples from 115 premenopausal women, 15 post-menopausal women and ectopic endometriotic lesions from 20 women with endometriosis. Participants/materials, setting, methods Full-thickness endometrium from hysterectomy tissues was analysed by immunohistochemistry for SSEA-1, SOX9 and β-catenin. Primary human endometrial epithelial cells from short-term cultures were sorted into SSEA1+/- fractions with a cell sorter or magnetic beads and analysed for markers of differentiation and pluripotency and telomere lengths (TLs) using qPCR, telomerase activity [telomere repeat amplification protocol (TRAP)] and growth in 3D culture. Main results and the role of chance Similar to the intestinal crypt epithelium, human endometrial basal glandular epithelial cells expressed nuclear SOX9 and contained a rare subpopulation of cells with nuclear β-catenin suggestive of an activated Wnt pathway. The embryonic stem cell-surface marker, SSEA-1, also marked the human endometrial basal glandular epithelial cells, and isolated SSEA-1(+) epithelial cells grown in monolayer showed significantly higher expression of telomerase activity, longer mean TLs, lower expression of genes for steroid receptors and produced a significantly higher number of endometrial gland-like spheroids in 3D culture compared with SSEA-1(-) epithelial cells (P = 0.009). Cells in ectopic endometriosis lesions also expressed SSEA-1 and nuclear SOX9, suggesting that the basalis contributes to ectopic lesion formation in endometriosis following retrograde menstruation. Limitations, reasons for caution This is a descriptive study with only short-term culture of the primary human epithelial cells in vitro. Wider implications of the findings The surface marker SSEA1 enriches for an endometrial epithelial cell subpopulation from the basalis. Since the functional endometrium originates from these cells, it is now possible to study basalis epithelium for stem/progenitor cell activity to extend our current understanding of endometrial biology in health and diseases. Study funding/competing interest(s) The work included in this manuscript was funded by Wellbeing of Women project grant RG1073 (D.K.H. and C.G.). We also acknowledge the support by National Health and Medical Research Council, RD Wright Career Development Award 465121 and Senior Research Fellowship 1042298, and the Victorian Government's Operation Infrastructure Support Program to C.G. and MRC G0601333 to T.V.Z. All authors have no conflict of interest to declare. Trial registration number N/A.

Mild oxidative stress as exerted by culture of human WI-38 fibroblasts under 40% oxygen partial pressure blocks proliferation irreversibly after one to three population doublings. Hyperoxically blocked cells are similar to senescent ones in terms of general morphology and lipofuscin accumulation. Moreover, they, like senescent fibroblasts, are blocked preferentially in G1 as evident from DNA content measurements by flow cytometry. Southern blotting of AluI- and HinfI-restricted genomic DNA shows an increase of the rate of telomere shortening from 90 bp per population doubling under normoxia to more than 500 bp per population doubling under hyperoxia. In every case, proliferation is blocked if a telomere cutoff length of about 4 kb is arrived at. The fact that telomere length correlates with the final inhibition of proliferation under conditions of varied oxidative stress, while the population doubling level does not, suggests that telomere shortening provides the signal for cell cycle exit in senescence. In postmitotic cells, no further telomere shortening occurs. However, the sensitivity of terminal restriction fragments to S1 nuclease increases, indicating the accumulation of single-strand breaks in telomeres of nondividing fibroblasts. This effect is found both under normoxic and hyperoxic culture, although it is more pronounced under conditions of higher oxidative stress. It might be speculated that accumulation of single-strand breaks and the resultant loss of distal single-stranded fragments during replication could be a major cause of telomere shortening, possibly more important than incomplete replication per se.

Lipofuscin accumulates in postmitotic cells. In human fibroblasts, accumulation of lipoftiscin as measured by cellular autofluorescence is exponential at first, but stops at a certain level. At the same time, cell death starts to decrease cell numbers significantly. Artificial lipofuscin-like material can be prepared by UV-crosslinking of mitochondria] preparations. This material is easily phagocytosed by human fibroblasts and results in an increased cellular lipofuscin accumulation. Such a forced lipofuscin accumulation is sufficient to block cellullar proliferation within a short time and to induce cell death as soon as the cellular lipofuscin autofluorescence reaches the same upper limit as that observed in senescent cells. It is concluded that accumulation of lipofuscin in postmitotic cells is not just an innocent consequence of ageing, but rather one amongst the important causes of senescence.

Element concentrations and local water content were measured in cytoplasm, nuclei, mitochondria and lipofuscin granula (LG) of isolated cardiac myocytes of the rat. Cells were cultured for 14 days under either 5%, 20% or 40% ambient oxygen concentration. LG were found to contain less cations and more phosphorous than mitochondria, which might be related to their lower protein and higher lipid content. Under oxidative stress, dehydration of mitochondria occurred, while their cation content remained constant. This is the same pattern of changes as found in cells in situ of aging rats. Therefore, it is concluded that peroxidative damage via oxygen derived free radicals is the reason for the mitochondrial water loss in the process of aging.

Fe2+/ascorbate-induced peroxidation of isolated rat liver mitochondria leads to initial volume changes and, ultimately, to severe damage characterized by gross swelling and loss of cristae and matrix material. Only the last phase is associated with significant production of malondialdehyde. The shrinkage of mitochondria during the onset of peroxidation matches changes observed in mitochondria of aging animals. Thioctacid (alpha-lipoic acid) prevents this initial shrinkage. However, its main effect in the system studied here is inhibition of active respiration.

BACKGROUND To test our hypothesis that eutopic secretory phase endometrium from women with endometriosis is similar to proliferative phase endometrium from fertile women without endometriosis, we explored the expression of regulators of cell fate across the menstrual cycle. METHODS Endometrial biopsies were taken from 73 women, comprising 38 women with surgically diagnosed active peritoneal endometriosis (Group 1) and 35 fertile women without endometriosis (Group 2). Nucleolin, proliferating cell nuclear antigen (PCNA), telomerase and histone γ-H2AX expression was evaluated by immunohistochemistry and mean telomere length (TL) by quantitative PCR. RESULTS We have immunolocalized nucleolin and γ-H2AX in the benign premenopausal endometrium for the first time. All markers were present in the proliferative phase endometrium of all women. In Group 2, during the secretory phase, proliferative markers declined with a paradoxical increase in stromal γ-H2AX. Women in Group 1, however, showed a persistent immunoreactivity for the proliferative markers, while the staining for γ-H2AX decreased in secretory endometrium (P < 0.05). This difference between groups was significant in both stroma and glands for nucleolin (P < 0.0001), PCNA (P < 0.01) and γ-H2AX (P < 0.05) in the secretory phase. We showed a positive correlation between mean TL and nucleolin expression (glandular r = 0.37, P = 0.002; stromal r = 0.4, P = 0.001), telomerase immunoreactivity (glandular r = 0.33, P = 0.009; stromal r = 0.4, P = 0.001) and glandular PCNA (r = 0.35, P = 0.004), whereas a negative correlation was seen between mean TL and γ-H2AX (r = −0.28, P = 0.04). CONCLUSIONS These findings demonstrate that the state of replication seen in secretory phase endometrium from women with active peritoneal endometriosis is not a simple extension of the proliferative phase.

BACKGROUND: In order to test our hypothesis that endometriosis is associated with abnormal expression of telomerase and telomere lengthening in endometrium, we assessed endometrial expression of the human telomerase enzyme and telomere length (TL). METHODS: This prospective pilot study, included 29 women with symptomatic, surgically diagnosed endometriosis (Group 1) and 27 healthy, fertile, symptom-free women without endometriosis (Group 2, confirmed by laparoscopy). Seventeen women in Group 1 and 15 women in Group 2 had endometrial biopsies taken on Day 21+ +2 of the cycle. A further 12 women in each group were biopsied on Day 26+2. Telomerase and estrogen receptor beta (ERb) expression was evaluated by immunohistochemistry. Mean TL was determined by quantitative PCR. RESULTS: The endometria of fertile healthy women showed either weak or no telomerase immunoreactivity throughout the luteal phase. Immunostaining for telomerase was significantly increased during the implantation window and the premenstrual endometria of women with endometriosis ( P< 0.0001). This was associated with a loss of stromal and vascular ERb immunostaining ( P< 0.05). The mean TL were significantly longer in endometria of women with endometriosis during the implantation window (P 5 0.005), indicating the biological relevance of our novel finding of telomerase in benign endometrium. There was positive correlation of the circulating estradiol with peripheral blood TL in women. CONCLUSIONS: We speculate that aberrant endometrial expression of telomerase mediates alterations in cell fate that enhance proliferation, contributing to the pathogenesis of endometriosis.

It has been established that telomere-dependent replicative senescence of human fibroblasts is stress-dependent. First, it was shown that telomere shortening, which is a major contributor to telomere uncapping, is stress-dependent to a significant degree. Second, the signalling pathway connecting telomere uncapping and replicative senescence appears to be the same as the one that is activated by DNA damage: uncapped telomeres activate signalling cascades involving the protein kinases ATM, ATR and, possibly, DNA-PK. Furthermore, phosphorylation of histone H2A.X facilitates the formation of DNA damage foci around uncapped telomeres, and this in turn activates downstream kinases Chk1 and Chk2 and, eventually, p53. It appears that this signalling pathway has to be maintained in order to keep cells in a senescent state. Thus, cellular senescence can be regarded as a permanently maintained DNA damage response state. This suggests that antibodies against DNA damage foci components might be useful markers for senescent cells in vivo.

Replicative senescence is characterized by the irreversible loss of division potential of cultivated human and animal cells. Correlations between the replicative potential in vitro and the age of the donor or the maximal lifespan of the species suggest replicative senescence to be an appropriate model for aging. Telomeres of human somatic cells shorten with each cell division but are stabilized at constant length in tumors and immortal cells by the enzyme telomerase. The assumption of a causal role of telomere shortening for the limited lifespan of cells in vitro was borne out recently. We could demonstrate oxidative stress as a main reason for telomere shortening. Telomeres are sensors for oxidative damage in the genome. Telomeres shorten during in vivo aging as well; however, there are significant differences between individuals. Telomere erosion might play a major role for the aging of the immune system. Our data suggest that telomere shortening in vivo could reflect the cumulative amount of oxidative damage to the organism. It might be useful as a biomarker of aging.

Telomere length in MRC-5 fibroblasts remains constant if the cells are proliferation-inhibited for up to 3 months by confluency. However, the apparent frequency of single-stranded sites in telomeres, measured as sensitivity to degradation by S1 nuclease, increases about fourfold during this extended inhibition of proliferation. After release of the cells, the frequency of telomeric single-stranded sites decreases to control values, and the telomere shortening rate increases about threefold as compared to controls proliferating without inhibition. This acceleration is transitory, the telomere shortening rate decreases to control values after about two population doublings after release. Finally, temporarily arrested fibroblast populations senesce at a lower cumulative population doubling level, but at about the same telomere length, as continuously proliferating controls. The data suggest that metabolic time-dependent single-strand degradation is a major cause of telomere shortening. They support the idea that telomere shortening plays an important role in triggering cellular senescence.

Telomeres, the nucleoprotein structures at the ends of all eukaryotic chromosomes, shorten with every cell cycle. Telomere shortening down to a critical level is believed to trigger proliferative senescence [1,2]. In contrast, germ line cells and cancer cells do not show telomere shortening. The ribonucleoprotein telomerase, a RNA-dependent DNA polymerase, maintains telomere length in immortal cells and might be essential for unlimited growth of cancer cells. Thus, telomerase could be an important target for anti cancer therapy.

Introduction Cross-sectional adult studies report associations between short leukocyte telomere length (LTL) and measures of vascular and cardiac damage. However, due to high inter-individual differences in LTL at birth and thereafter, it is unclear whether this relationship is due to inherited short telomere length or a higher rate of LTL shortening through life. In this study we explored whether the baseline LTL or the rate of LTL shortening could predict cardiovascular (CV) phenotypes during 10 year follow up in the MRC National Survey of Health and Development (NSHD, also known as 1946 birth cohort) study. Methods 689 participants with measures of LTL and CV risk factors at 52 and 62 years underwent measurements of right common carotid intima-media thickness (cIMT), cardiac mass and left ventricular ejection fraction at 62 years. LTL was measured by real time PCR. Results At both time points a negative association was found between LTL and age (p Conclusions/implications This is the first study showing an association between rate of LTL attrition and final vascular phenotype. These findings suggest that the rate of progression of cellular aging in late midlife (reflected by the rate of LTL attrition) influences predisposition to vascular damage, over and above the contribution of CV risk factors. Therefore, current results identified a possible novel pathway associated to an increased risk of CV disease. This can possibly improve the CV risk stratification of the patients. Furthermore, a better understanding of the factors influencing the rate of LTL has the potential to identify novel terapeutic targets and lead to a more effective CV disease prevention.

Uptake of cadmium into cultured cells and its effects on cell growth and DNA synthesis are measured over a range of Cd concentrations of seven orders of magnitude. Cd uptake is found to be proportional to the external Cd concentration and to incubation time over a very broad range of concentrations. At least 200 mmol cadmium per kg dry weight of cells can be accumulated in this way, leading to exhaustion of the major intracellular Cd binding sites before cell death. On the other hand, very low cadmium concentrations down to 100 pM stimulate cell growth and DNA synthesis significantly. Stimulation is found in all three mammalian cell types examined: namely L6J1, a rat permanent myoblast cell line, LLC-PK1 porcine renal epithelial cells, and a primary rat chondrocyte culture. Cd acts as a cofactor with serum in L6J1 cultures, but is stimulatory only in serum-free cultures of chondrocytes. Stimulation occurs at Cd concentrations too low to result in a measurable induction of metallothionein. This might implicate the action of response amplifiers in the chain of events leading to Cd-stimulated DNA replication and cell growth.

Worldwide, the oldest old are the fastest growing section of the population yet there is little up-to-date information about their health or factors which maintain health and independence. The Newcastle 85+ Study is exploring the spectrum of health within an inception cohort of 85 year-olds and examining health trajectories and outcomes as the cohort ages. Health status at baseline will be presented. Members of the 1921 birth cohort were recruited from general practices in Newcastle and North Tyneside, …

SUMMARY The proposed cryofixation technique uses a tubule-shaped needle chilled in liquid propane for simultaneous excision and freezing of a tissue specimen. Due to this simultaneity, ionic shifts created by traumatic influences are avoided even in the outermost cells of the specimen. Moreover, it is shown here that stopping the blood flow for more than about 10 s results in notable ionic shifts between cells and extracellular space in rat heart and liver. Such preparative ischaemic injury is minimized by the Fast Cryofixation Technique because it can be easily performed on organs within the circulatory system, whilst the heart of the animal is still beating. Intracellular concentrations of the monovalent ions in rat heart and liver, obtained by this method, tally well with recent results from different independent techniques reported in the literature. As demonstrated by cross-sectioning and freeze-fracturing, the structural preservation of the freezing technique is sufficient for X-ray microanalytical work.

Age-dependent changes of the intracellular water content (IWC) of the rat myocardium have been measured by X-ray microanalysis of deep-frozen bulk specimens (Zs.-Nagy et al., 1982), using a slow warming up and drying of a very superficial layer of the sample, in order to minimize space charging effects. These results were compared with those calculated from the conventionally measured tissue water contents (TWC) and data from literature of morphologically estimated volume density of the extracellular space (David et al., 1981). The IWC values obtained from these two independent methods are in very good agreement, showing that the etching of the surface during the bulk specimen analysis is sufficiently small, i.e., it does not result in any considerable error in the quantitative X-ray microanalysis. The IWC of heart muscle cells decreases significantly with advancing age (from about 80% by weight at the age of 14 days to 71% by the age of 24 months). This observation is consistent with the membrane hypothesis of aging (Zs.-Nagy, 1978). Comparison of the IWC with TWC of the heart muscle shows that the increase of the volume density of the extracellular space during aging balances the age-dependent loss of myocytes to a certain extent.

The precipitation patterns of the following ultracytochemical methods in rat muscle cells were compared and examined critically: the potassium pyroantimonate method for calcium demonstration; the calcium phosphate technique for the Ca2+--ATPase reaction; the formazan reaction for the demonstration of creatine kinase activity (all performed on heart muscle); and the lead phosphate technique for the Mg2+--ATPase reaction in skeletal muscle. Using X-ray microanalysis, it was found that the antimonate precipitate contains only calcium as the precipitated ion in the vast majority of cases. Most probably it consists of pure calcium pyroantimonate. However, in myocytes showing the well-established precipitation pattern, the concentration of calcium was estimated to be about two orders of magnitude higher than the native concentration of total intracellular calcium. It is concluded that calcium ions diffuse freely from the extracellular space and from adjacent cells into cells containing antimonate and are precipitated mostly at sites where heterogeneous nucleation is facilitated by intracellular catalysts (biopolymers). As shown by the similar precipitation patterns for the four reactions compared, these catalysts are not specific to any of these reactions and are most probably neither calcium-binding sites nor sites of any one of the enzymes examined in the native cell.

Elemental concentrations in different compartments of cryosections of isolated rat liver cells cryotransferred and freeze-dried were compared with those obtained after storage under vacuum for 12 or 60 h and after exposure to room air for 2 min. Poorer image contrast and segregation artefacts are frequently found in air-exposed sections, together with a slight but significant decrease of the K concentration in the cytoplasm and an increase of the S concentration in the liver cell nuclei and the extracellular medium. Extreme distortions of both ultrastructure and elemental distributions are observed if the sections are even slightly colder than the surrounding atmosphere. While storage of frozen-dried cryosections under vacuum for less than 12 h does not lead to alterations in the sections, gross changes are found both in morphology and elemental distribution in sections stored under vacuum for about 60 h. Long-time vacuum storage of frozen-dried cryosections is, therefore, not recommended.