Tissues Of Old Animals Research Articles

Dynamic Changes in the Activity and Content of Particular Proteasome Forms in Cerebral Cortex of C57BL/6 Mice during Aging

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes were revealed, as well as an increased expression of their individual forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in the cerebral cortex samples from C57BL/6 mice at the age of 60, 190, 380, and 720 days. In addition, the content of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis under native conditions were characterized. Compared with samples from young animals, in the cerebral cortex of animals aged 720 days the following changes in the expression patterns of proteasome genes were revealed: a decrease in the expression of PSMB5 gene encoding the constitutive proteasome subunit β5; activation of genes encoding immune subunits β5i and β1i. In clarified tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, decreased chymotrypsin-like activity and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity were also revealed. Analysis of the activity of native complexes in the tissues of old animals revealed decreased chymotrypsin-like activity of both 26S and 20S proteasomes containing the β5i subunit. Based on the data obtained, it can be assumed that changes in the pool of non-constitutive proteasomes reflect aging-associated adaptive processes in mouse brain.

Umbilical cord plasma concentrate has beneficial effects on DNA methylation GrimAge and human clinical biomarkers.

Plasma transfusions are standard treatments to replace missing proteins in people with rare genetic diseases. Prior studies have demonstrated that heterochronic parabiosis has beneficial effects on several tissues of old animals receiving young blood. Human clinical trials are currently underway to investigate whether the infusion of plasma or plasma‐derived factors from young donors can be used to mitigate human age‐related conditions. Here, we use data from a safety study (n = 18, mean age 74) to investigate whether human umbilical cord plasma concentrate (hereinafter Plasma Concentrate) injected weekly (1 ml intramuscular) into elderly human subjects over a 10‐week period affects different biomarkers, including epigenetic age measures, standard clinical biomarkers of organ dysfunction, mitochondrial DNA copy number (mtDNA‐CN), and leukocyte telomere length. This study shows that treatment with plasma concentrate is safe. More than 20 clinical biomarkers were significantly and beneficially altered following the treatments. For example, creatinine was significantly decreased (p = 0.0039), while estimated glomerular filtration rate (eGFR) was increased (p = 0.0044), indicating the treatment may improve biomarkers of kidney function. Three of four immunoglobulin biomarkers decreased, while telomere length and mtDNA‐CN were not significantly affected by the treatment. The treatment reduced DNA methylation‐based GrimAge by an average of 0.82 years (p = 0.0093), suggests a reduction in morbidity and mortality risk. By contrast, no significant results could be observed for epigenetic clocks that estimate chronological age. Our results support the view that plasma concentrate contains youth‐promoting factors.

Stimulation of the endogenous hydrogen sulfide synthesis restores constitutive nitric oxide synthesis and endothelium-dependent vasodilation in old rats

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Hydrogen sulfide (H2S) is a gas transmitter of endogenous origin, which has an important regulatory function in the human body. Endogenous H2S is synthesized by three enzymes, cistationine-β-synthase (CBS), cistationine-γ-liase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MTS), which is coupled to the activity of cysteine aminotransferase (CAT). CAT, CBS, and CSE are pyridoxal-5- phosphate (PLP)-dependent enzymes. It is known that with age the exchange of hydrogen sulfide is disturbed against the background of increasing oxidative and nitrosative stress. At the same time there is a decrease of constitutive synthesis of nitric oxide (NO). Recent studies show that hydrogen sulfide closely interacts with the NO system. Activation of H2S biosynthesis and restoration of its pools in tissues of old animals may be a promising direction of cardiovascular disease prevention. The aim of the current work was to study the effect of H2S-synthesizing enzymes cofactor pyridoxal-5-phosphate (PLP) on the endothelium-dependent relaxation of smooth muscle vessels in old rats, as well as the H2S content and cNOS activity in aortic tissues. Methods Old rats (22-24 months old) were PLP administered per os in dose of 0,7 mg per kg daily for 2 weeks.The content of hydrogen sulfide, activity of cNOS and iNOS was measured in aortic tissues.Vascular reactions were determined in experiments on isolated aortic strips. To clarify the role of either NO or H2S in the endothelium-dependent vasorelaxation, N-nitro-L-arginine methyl ester hydrochloride (L-NAME) (Sigma, USA, 27 mg/kg) or O-carboxymethylhydroxylamine (O-CMH) (50 mg/kg) (Sigma, USA) was injected intraperitoneally 30 min before sacrificing. Results We showed that the H2S content was 1.6 times lower in old rat aorta compared to adult ones. PLP administration for 2 weeks induced a significant increase of H2S content in aortic tissue of old rats (P<0.05). It was found that with aging, the activity of cNOS decreases by 3 times, and the stimulation of endogenous Н2S leads to an increase in the activity of this enzyme in the aortic tissues by 2.1 times as compared with those in old animals. At the same time, the activity of the iNOS enzyme with aging increases by 3.8 times, and after the introduction of PLP, it decreases almost halved. Also shown, that PLP significantly improved endothelium-dependent aortic smooth muscle relaxation in old rats. The amplitude of acetylcholine-induced relaxation which was significantly reduced in old rats was increased more than 2.5-fold. This effect was reversed by inhibitors of NO-synthase and 3-mercaptopyruvate sulfurtransferase indicating the involvement of NO and H2S in the improved endothelium-dependent vascular relaxation. Conclusions Thus, H2S acts as a regulator of the NO system that increases cNOS activity and NO synthesis in aortic tissue, which improves endothelium-dependent relaxation of aortic smooth muscle in aged rats.

Investigation of the effects of aging on the expression of aquaporin 1 and aquaporin 4 protein in heart tissue.

Objective:Aquaporin (AQP) 1 and AQP 4 are expressed in human heart and several studies have been focused on these two aquaporins. For this purpose, the present study is aimed to research the effects of aging on AQP 1 and AQP 4 in heart tissue.Methods:In this study, 14 Balb/C type white mice were used. Animals were divided into two equal groups. Group I consisted of 2-month-old young animals (n=7), and group II consisted of 18-month-old animals (n=7). To determine the AQP1 and AQP4 expression in the myocardium, the heart tissue was removed to perform western blotting and immunohistochemical and histopathological evaluations.Results:Muscle fibers of the heart in aged animals were more irregular and loosely organized in hematoxylin–eosin (H&E) stained sections. H-score analysis revealed that AQP1 and AQP4 immunoreactivity significantly increased in heart tissues of old mice compared with those of young mice (p<0.001). In addition, AQP1 and AQP4 protein expressions in the tissues of old animals were increased significantly according to western blot analysis (p=0.018 and p<0.001 for AQP1 and AQP4, respectively).Conclusion:Increased AQP1 and AQP4 levels in the heart tissue may be correlated with the maintenance of water and electrolytes balance, which decreases with aging. In this context, it might be the result of a compensatory response to decreased AQP4 functions. In addition, this increase with aging as demonstrated in our study might be one of the factors that increases the tendency of ischemia in elder people.

Age related changes in selenium and glutathione levels in different lobes of the rat prostate

Aging represents a major risk factor for prostate cancer; however, mechanisms responsible for this relationship remain unclear. Preclinical and some clinical investigations support the protective role of selenium against prostate cancer possibly through the reduction of oxidative stress. While increased levels of oxidative stress together with decreases in selenium and the major cellular antioxidant glutathione (GSH) are common in tissues of old animals, there is little data available on these parameters in the prostate. In the present study we have compared the levels of selenium, GSH and protein-bound GSH (GSSP) in blood and prostate tissues in young (4-month), mature (12-month), old (18month), and very old (24month) male F344 rats. Each prostate lobe (dorsolateral, DL; anterior, AL; ventral, VL) was analyzed separately based upon their differing potential for prostate cancer development. At all ages, selenium levels were lowest in DL<VL<AL. After 12mo, an 85% reduction in selenium in the DL was observed (P<0.05), while levels in other lobes were unchanged. In animals of all ages, levels of GSH were lowest in the VL<DL=AL and no significant changes were observed in GSH levels by 18 mo. However, GSSP, a marker of oxidative stress, was increased 90% after 18mo in the DL only (P<0.01). These findings of age-related changes in GSSP and selenium in the DL prostate are consistent with the sensitivity of this lobe to carcinogenesis and, thus, may be playing a mechanistic role.

Age-associated loss in adiponectin-activation by caloric restriction: Lack of compensation by enhanced inducibility of adiponectin paralogs CTRP2 and CTRP7

Hormonal signals from adipose tissue regulate energy homeostasis but may also be involved in the anti-aging effects of caloric restriction. The purpose of the current study was the investigation of age-dependent effects of caloric restriction on the release of adiponectin, on the expression and activation of adiponectin-related signaling and on parameters of altered insulin sensitivity. In young and in senescent rats, 2 months moderate caloric restriction reduces serum leptin and insulin (young: −50%; old: −30%) suggesting increased insulin sensitivity. However, the same diet enhances serum adiponectin in young (+60%) but not in senescent (+2%, n = NS) rats. Similarly, adiponectin expression (visceral fat) and muscular AdipoR1/2 expression are induced in young rats but not in senescent rats. The locally produced adiponectin paralogs CTRP2/7 are elevated in muscular tissues of old animals (CTRP2 protein: +40%; CTRP7 protein: +50%) and further induced by caloric restriction but this does not result in an increased activation of their downstream target AMPK. Thus, aging is associated with a partial loss of adiponectin inducibility following moderate caloric restriction. This loss is not sufficiently compensated by the locally induced adiponectin paralogs CTRP2/7, although caloric restriction results in increased insulin sensitivity in young and in senescent animals. Thus, the improvement in insulin sensitivity appears to be independent of adiponectin induction by caloric restriction in this model.

Accumulation of Dolichol in Older Tissues Satisfies the Proposed Criteria To Be Qualified a Biomarker of Aging

Criteria for defining biomarkers have been suggested. Accumulation of dolichol in tissues of older animals meets the following criteria: (a) levels of dolichol exhibit a quantitative correlation with age in all tissues and are not altered by several age-dependent diseases in the same direction as that of aging; (b) accumulation is not secondary to metabolic changes of aging and is altered appropriately by factors that modulate the aging rate like caloric restriction and physical exercise; (c) biomarker is applicable to different tissues across mammalian species, including humans, and to trisomy 21 and its hypothalamic digoxin-mediated model. Reliable changes in tissue dolichol levels are seen in relatively short intervals of time compared to over a life span, and levels can be tested on a small amount of tissue without causing death of the animal. In this article, we show applications to the study of host-graft interaction and detection of gender-related differences in biological age, and we discuss mechanism(s) of accumulation.

Age-related changes in neuronal uptake of noradrenaline

Age-related changes in the neuronal uptake of noradrenaline were studied in the vas deferens of 4-, 12- and 20-months old rats by determining the potentiating effect of cocaine on concentration-effect curves to noradrenaline and by evaluating the kinetic parameters (Vmax and apparent Km) of neuronal 3H-noradrenaline uptake. Since the neurotransmitters released by nerve terminals and the density of noradrenergic innervation are different in the epididymal and prostatic portion of the vas, the experiments were carried out in these two portions of the organ. In the epididymal portion, the ratio between EC50 for noradrenaline in the presence and in the absence of cocaine was reduced in tissues of middle-aged (12-months) and old (20-months) animals when compared with young (4-months) animals. The pD2 values for noradrenaline in the absence of cocaine and the kinetic parameters for neuronal 3H-noradrenaline uptake were not modified by ageing. Thus, in the epididymal portion there was no age-related change in neuronal uptake. The age-related decrease in the cocaine-induced supersensitivity to noradrenaline was due to an age-related decrease in the pD2 value for noradrenaline observed in the presence of cocaine, suggesting postsynaptic changes. In the prostatic portion, the ratio between EC50 for noradrenaline in the presence and in the absence of cocaine was also reduced in tissues of old animals (20-months) when compared with middle-aged and young animals. The pD2 value for noradrenaline in the absence of cocaine obtained in the prostatic portion of 20-months old rats was higher than that in the younger groups (4- and 12-months).(ABSTRACT TRUNCATED AT 250 WORDS)

Age-associated accumulation of altered FDP aldolase B in mice: Conditions of detection and determination of aldolase half life in young and old animals

Inactive enzyme molecules in aging animals have been described for mouse aldolase B [l] and aldolase A [2], nematode aldolase [3,4], and other enzyme systems [5-Q We have postulated that the accumulation of inactive enzyme molecules in cells of aging organisms could be due, at least in part, to their inefficient disposal by the protein degradation system [7,9 1. In [lo] no difference in the activity of aldolase B from young or old mice was observed. It was suggested that our results with aldolase B [ 11, and possibly other results concerning age-related inactive enzyme molecules, were due to an artifact emanating from increased proteolysis and possible removal of the COOH-terminal tyrosine in enzyme preparations from tissues of old animals. Here we show that when liver homogenates are carefully prepared in isotonic buffers and lysosomes are removed (as is routinely done in our laboratory) no loss of enzyme activity due to proteolysis occurs. Under these conditions, freshly prepared homogenates of livers of old animals contain aldolase with reduced catalytic activity/unit of enzyme antigen. This observation fully corroborates [ 11. We also describe here an increased t,,s of aldolase B in livers of old animals. This increase at least can partially explain the basis for the accumulation of inactive enzyme molecules in livers of old animals.

On the age-induced antigenic peculiarities of tissues in old animals

An experimental work was carried out to determine the antigenic properties of blood sera and of the saline extracts from animal tissues (cows and albino rats) of a different age. As established, blood sera and saline extracis from the organs and tissues of old animals, apart from the common antigenic properties to the analogous preparations from the young animals, possessed their own specific antigenic peculiarities, connected with the aging of the organism.

Antigenic features characteristic for lipid-polysaccharide complexes in the tissues of old animals

A number of experiments were conducted to detect the antigenic features of lipid-polysaccharide complexes in the tissues of animals of various age. The lipid-polysaccharide complexes from the animals' tissue are complete antigens; the complexes mentioned have specific antigenic peculiarities connected with age.

THE LIQUID MEDIUM IN TISSUE CULTURE

SummaryIn the past, largely on an empirical basis, it was found that the most effective liquid media for culturing animal tissue cells consisted of mixtures of physiological saline solutions, tissue extracts (preferably from embryonic tissue) and serum or plasma. The marked influx of investigators with primarily biochemical interests into the tissue culture field in the last few decades has led to determined efforts to learn more of the precise relationships between the composition of these media and the metabolism of the tissues. Since both of these are so complex, and the equilibrium between them is such a delicate one, it is perhaps not surprising that many apparently contradictory results are seen in the current literature.The place of physiological salt solutions in tissue cultures appears to be a relatively simple matter, and their functions are reasonably well understood. The present confusion relates to the role of the tissue extract and serum components of the medium. For certain types of cells there is evidence that a tissue‐extract nucleo‐protein fraction, supplemented by a surprisingly small group of smaller molecule nutrients, is adequate to support growth, at least for short periods. Various possible mixtures of these smaller components may be effective as a supplement in the same system, although it is probable that optimum combinations exist, again depending on the cell type and the culturing conditions. The tissue nucleoprotein fraction cannot be considered as a ‘growth hormone’, in the conventional sense of that term, since other cell types, or even superficially similar cell types taken from other animal donors, are able to maintain themselves satisfactorily when this fraction is completely absent, at least if serum proteins are present in the medium. It is thus evident that quantitative comparisons of data found by various researchers are of very dubious validity unless it is certain that identical culturing techniques were applied, using identical cell systems.On a more positive note, however, it seems probable that the analytical approach to the liquid medium problem is bringing the long‐sought goal of a controlled composition ‘synthetic’ medium for cultured animal cells much nearer to attainment for certain systems sharply defined as to cell type and culturing procedures.There are also indications that ‘growth’, biochemically speaking, as defined by the cell division in a culture, may be a separate process from ‘growth’ as defined by increase in culture mass, protein content or similar criteria. In other words, the chemical and physical conditions required to aid in inducing or in completing the mitotic cycle are not necessarily directly related to the nutritional requirements of growing and maturing individual cells in a healthy culture.There is some evidence that extracts made from tissues of older animals are, in many culture systems, as effective as extracts of embryonic tissue. Since, on the whole, such tissues are more accessible, their wider use by tissue culturists should be investigated more extensively.The role of serum in culture media is still not clear. In some cases it is apparently not required at all, in others it has a beneficial effect. Many of the reported contradictions as to the value or lack of value of serum in culture media are probably related to its variability in composition and state of preservation, and to the age and health of the donor animal. Until more experiments are done with better defined materials, resolving these conflicting results does not appear to be possible.A synthetic medium which would either fully support growth and development of all tissues, or one which would allow growth and development in limited but defined systems, is still the most logical goal as a reference medium and as the starting point for the systematic study of all phases of culture behaviour. The progress toward this goal has been definite but limited. With the tools now available for fractionating biological mixtures, synthesizing compounds of biological interest, and elucidating mechanisms of metabolism, the realization of a completely controlled medium, and possibly a completely synthetic medium, appears now to be merely a matter of time and continued effort. The advances in knowledge of tissue growth and behaviour that have already been achieved in the absence of such a medium are very impressive, and a constantly accelerating rate of significant research results is to be expected as the interest in this field increases.