Association between subcutaneous and intramuscular fat content in porcine ham and loin depending on age, breed and FABP3 and LEPR genes transcript abundance.

The objective of the present study was to analyze the level of intramuscular fat (IMF) in loin (musculus longissimus dorsi) and ham (musculus semimembranosus) and the level of subcutaneous fat in these cuts depending on breed, age and the expression level of FABP3 and LEPR genes. The results obtained showed that only the breed influenced on the level of both intramuscular and subcutaneous fat to the same extent (P ≤ 0.001). The age of animals had an effect on fat content of the cuts (P ≤ 0.001) and to a lower extent on the level of IMF in both muscles (P ≤ 0.05). We confirmed highly significant effect of breed and age on the LEPR mRNA abundance--the expression of the this gene increased significantly (P ≤ 0.01) with age and the highest expression was found for the Pulawska breed in m. longissimus dorsi and for the Polish Landrace breed in m. semimembranosus. We observed the high correlations between the transcript level of the LEPR gene and the fat content of individual cuts (P ≤ 0.01). The expression level of FABP3 gene influenced the level of IMF (P ≤ 0.01), but not the level of subcutaneous fat in loin and ham.

Autophagy, organelles and ageing.

As a result of insufficient digestion of oxidatively damaged macromolecules and organelles by autophagy and other degradative systems, long-lived postmitotic cells, such as cardiac myocytes, neurons and retinal pigment epithelial cells, progressively accumulate biological 'garbage' ('waste' materials). The latter include lipofuscin (a non-degradable intralysosomal polymeric substance), defective mitochondria and other organelles, and aberrant proteins, often forming aggregates (aggresomes). An interaction between senescent lipofuscin-loaded lysosomes and mitochondria seems to play a pivotal role in the progress of cellular ageing. Lipofuscin deposition hampers autophagic mitochondrial turnover, promoting the accumulation of senescent mitochondria, which are deficient in ATP production but produce increased amounts of reactive oxygen species. Increased oxidative stress, in turn, further enhances damage to both mitochondria and lysosomes, thus diminishing adaptability, triggering mitochondrial and lysosomal pro-apoptotic pathways, and culminating in cell death.

Lysosomal labilization.

The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells.

Effect of the polymorphism of prolactin receptor (PRLR) and leptin (LEP) genes on litter size in Polish pigs.

The aim of the experiment was to use the DNA mutations in the PRLR and LEP genes to determine associations between the genotype and litter size in Polish Large White x Landrace sows. Reproductive traits investigated were: total number of piglets born (TNB), number of piglets born alive (NBA) and number of piglets weaned. The polymorphism in PRLR and LEP genes was detected using the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method, with specific primers and the restriction enzymes AluI and HinfI respectively. Two different alleles of PRLR and LEP gene were identified: alleles A (0.62) and B (0.38) of the PRLR gene and alleles C (0.10) and T (0.90) of the LEP gene. The relationships between the PRLR and LEP genotypes and TBN, NBA and NW were analysed. The analysis showed, in first parity sows, statistically significant (p < or = 0.01) differences between sows carrying different PRLR genotypes. In later parities, sows with the AA genotype still had the largest litter size compared with AB and BB sows, but the difference was statistically not significant. Analysis of the interaction PARITY x PRLR showed small and statistically not significant differences. The analysis of relationship between different LEP genotypes and TNB, NBA, NW showed small and statistically non-significant differences.

Vitamin E analogues as inducers of apoptosis: implications for their potential antineoplastic role.

Recent evidence suggests that vitamin E and its analogues, which have been used for many years as antioxidants, may not only protect cells from free radical damage but also induce apoptotic cell death in various cell types. While alpha-tocopherol (alpha-TOH) is mainly known as an anti-apoptotic agent, its redox-silent analogues either have no influence on cell survival (alpha-tocopheryl acetate, alpha-TOA), or induce apoptosis (alpha-tocopheryl succinate, alpha-TOS). Although precise mechanisms of apoptosis induction by alpha-TOS remain to be elucidated, there is evidence that this process involves both the antiproliferative and membrane destabilising activities of the agent. Alpha-TOS has been shown to induce apoptosis in malignant cell lines but not, in general, in normal cells, and to inhibit tumorigenesis in vivo. These features suggest that this semi-synthetic analogue of vitamin E could be a promising antineoplastic agent.

Garbage catastrophe theory of aging: imperfect removal of oxidative damage?

Increasing evidence suggests an important role of oxidant-induced damage in the progress of senescent changes, providing support for the free radical theory of aging proposed by Harman in 1956. However, considering that biological organisms continuously renew their structures, it is not clear why oxidative damage should accumulate with age. No strong evidence has been provided in favor of the concept of aging as an accumulation of synthetic errors (e.g. Orgel's 'error-catastrophe' theory and the somatic mutation theory). Rather, we believe that the process of aging may derive from imperfect clearance of oxidatively damaged, relatively indigestible material, the accumulation of which further hinders cellular catabolic and anabolic functions. From this perspective, it might be predicted that: (i) suppression of oxidative damage would enhance longevity; (ii) accumulation of incompletely digested material (e.g. lipofuscin pigment) would interfere with cellular functions and increase probability of death; (iii) rejuvenation during reproduction is mainly provided by dilution of undigested material associated with intensive growth of the developing organism; and (iv) age-related damage starts to accumulate substantially when development is complete, and mainly affects postmitotic, cells and extracellular matrix, not proliferating cells. There is abundant support for all these predictions.

Ceroid/lipofuscin-loaded human fibroblasts show increased susceptibility to oxidative stress.

To test whether the possibly enhanced sensitivity of aged cells to oxidative stress may depend on their content of ceroid/lipofuscin, AG-1518 human fibroblasts with various amounts of the pigment accumulated due to prolonged cultivation under normobaric hyperoxia were exposed to acute oxidative stress (2.5 microM naphthazarin, 15 min) and then returned to standard culture conditions. Twenty-four hours after the naphthazarin treatment, 37% of the cells were still vital, whereas others had undergone oxidative stress-induced apoptosis with ensuing postapoptotic necrosis. The average amount of ceroid/lipofuscin within the surviving cells was only about half of that of the initial population of cells, as measured before the naphthazarin exposure. This finding suggests that ceroid/lipofuscin-rich cells have an increased sensitivity to oxidative stress. The ceroid/lipofuscin quantity strongly positively correlated with the size of the acidic compartment (as evaluated by uptake of the weakly basic lysosomotropic fluorochrome acridine orange) and with its content of the lysosomal protease cathepsin D, as assayed by immunocytochemistry. We hypothesize that the enhanced sensitivity of ceroid/lipofuscin-loaded cells to oxidative stress may be caused by the increased amounts of lysosomal enzymes, known as mediators of oxidative damage, and/or by catalysis of intralysosomal oxidative reactions by lipofuscin-associated iron.

Ceroid/lipofuscin-loaded human fibroblasts show decreased survival time and diminished autophagocytosis during amino acid starvation.

To test whether heavy accumulation of ceroid/lipofuscin can disturb important functions of the lysosomal system, AG-1518 human fibroblasts, ceroid/lipofuscin-loaded (following prolonged culture at normobaric hyperoxia) or not, were exposed to amino acid starvation. Ceroid/lipofuscin-loading resulted in decreased cellular survival. Also, there was an inverse relationship between amounts of ceroid/lipofuscin and the survival time of individual cells within the same cultures. Ceroid/lipofuscin-loaded fibroblasts displayed diminished autophagocytotic capacity, as demonstrated by electron microscopy and by treatment of cell cultures with NH4Cl (which inhibits autophagocytotic degradation by increasing intralysosomal pH) for 1 week before ensuing starvation. The latter treatment increased survival of control cells (due to deposition of nondegraded autophagocytosed material before start of starvation), but not that of ceroid/lipofuscin-loaded cells. Moreover, when NH4Cl treatment was combined with starvation, both groups of cells showed approximately the same shortened survival times, testifying to the causal relationship between diminished autophagocytosis and decreased survival of starving ceroid/lipofuscin-loaded cells. We hypothesize that large amounts of undegradable ceroid/lipofuscin within the acidic vacuolar compartment may interfere with lysosomal function, resulting in poor renewal of long-lived proteins and worn-out/damaged organelles, decreased adaptability, and cell death.

Ceroid/lipofuscin formation in cultured human fibroblasts: the role of oxidative stress and lysosomal proteolysis.

The mechanisms involved in the accumulation of ceroid/lipofuscin within non-dividing cells are not totally understood. Oxidative stress, as well as diminished activity of lysosomal proteolytic enzymes, are known to induce ceroid/lipofuscin accumulation in a variety of cell types. In order to clarify the roles of oxidative stress and lysosomal proteolysis in ceroidogenesis/lipofuscinogenesis, and to study the fate of already formed ceroid/lipofuscin, confluent cultures of AG-1518 human fibroblasts were exposed to oxidative stress (40% ambient oxygen) and/or treated with the thiol protease inhibitor leupeptin for 2 weeks. Both oxidative stress and protease inhibition caused accumulation of ceroid/lipofuscin per se (estimated by fluorescent, confocal and electron microscopy). The combined effect of these factors was, however, almost three times as large as the sum of their isolated effects. The pigment accumulated progressively as long as the oxidative stress and/or protease inhibition acted; was not eliminated after re-establishment of normal conditions; and decreased in amount after subsequent passage. The results suggest that (i) ceroid/lipofuscin forms within secondary lysosomes due to peroxidative damage of autophagocytosed material, and (ii) it is not substantially eliminated from non-dividing cells by degradation or exocytosis.

On the degradability and exocytosis of ceroid/lipofuscin in cultured rat cardiac myocytes.

The accumulation of lipofuscin (LF)--a polymeric, electron-dense, autofluorescent substance--within postmitotic cells is a characteristic manifestation of aging. It is generally believed that LF is undegradable and formed due to peroxidative alterations of various macromolecules under intralysosomal autophagic degradation. We report here that a short-term exposure of cultured neonatal rat cardiac myocytes to the thiol protease-inhibitor leupeptin, causes an accumulation of numerous electron-dense autophagic lysosomes within the cells. Although very similar to LF by ultrastructure, these inclusions do not display LF-specific, yellow-orange autofluorescence when excited with blue light. Moreover, they rapidly disappear from the cells upon re-establishment of normal culture conditions. In contrast, prolonged leupeptin treatment results in an accumulation of dense lysosomes that also show LF-typical autofluorescence. This autofluorescent material remains in the cells after the end of leupeptin action. The results suggest that: (i) a certain amount of time is needed for autophagocytosed material to become peroxidized, autofluorescent and undegradable, i.e. to acquire properties typical of LF; (ii) protease-inhibition by itself does not lead to LF-formation but rather allows the prolonged time needed for oxidative modification of autophagocytosed material; (iii) mature LF is probably not subjected to either degradation or exocytosis.

Lipofuscin: mechanisms of formation and increase with age.

Lipofuscin (age pigment) is a brown-yellow, electron-dense, autofluorescent material that accumulates progressively over time in lysosomes of postmitotic cells, such as neurons and cardiac myocytes. The exact mechanisms behind this accumulation are still unclear. This review outlines the present knowledge of age pigment formation, and considers possible mechanisms responsible for the increase of lipofuscin with age. Numerous studies indicate that the formation of lipofuscin is due to the oxidative alteration of macromolecules by oxygen-derived free radicals generated in reactions catalyzed by redox-active iron of low molecular weight. Two principal explanations for the increase of lipofuscin with age have been suggested. The first one is based on the notion that lipofuscin is not totally eliminated (either by degradation or exocytosis) even at young age, and, thus, accumulates in postmitotic cells as a function of time. Since oxidative reactions are obligatory for life, they would act as age-independent enhancers of lipofuscin accumulation, as well as of many other manifestations of senescence. The second explanation is that the increase of lipofuscin is an effect of aging, caused by an age-related enhancement of autophagocytosis, a decline in intralysosomal degradation, and/or a decrease in exocytosis.

[The mechanisms of the increase in the amount of lipofuscin in the cells during aging]. / Mekhanizmy uvelicheniia kolichestva lipofustsina v kletkakh pri starenii.

The literary data concerning the mechanisms of an increase of lipofuscin amount in cells are reviewed. The paper contains information about the properties of lipofuscin inclusions, the influence of autophagocytosis, intralysosomal degradation, and exocytosis on lipofuscin content in cells. The question of the possibility of complete elimination of autophagocytosed material is discussed.

[The immunological and morphological aspects of hemoperfusion with pig donor spleen in treating psoriasis patients]. / Immunologicheskie i morfologicheskie aspekty gemoperfuzii donorskoi selezenki svin'i pri lechenii bol'nykh psoriazom.

The present paper focuses on the immunological and morphological aspects of extracorporal joining up of donor porcine spleen in the treatment of psoriasis. It has been ascertained that extracorporal joining up of donor porcine in the treatment of psoriasis makes for normalization of bodily immunologic reactivity leading to regression of psoriatic eruptions.

The effect of age on formation and elimination of autophagic vacuoles in mouse hepatocytes.

To gain an understanding of the mechanisms of lipofuscin (LF) accumulation in aging cells we studied both the formation and the elimination of autophagic vacuoles (AV) in hepatocytes of adult (5-6 months) and old (20-21 months) CBA male mice. For the evaluation of AV formation we determined the degree of AV accumulation 4 h after the injection of vinblastine, which blocks the fusion of AV with lysosomes. Other animals were treated with Triton X-100, which provokes the appearance of AV, and then, after 4 h, with cycloheximide to block the appearance of newly formed AV. AV elimination was then evaluated during the next 30 minutes. In our quantitative electron microscopic study of the liver tissue of these mice, we found a decrease in AV formation rate as well as a decrease in the intensity of AV elimination in hepatocytes of old versus young adult animals. The results indicate that the slowing down of AV elimination with age may play an important role in LF accumulation. Further, LF accumulation manifests itself despite decreased autophagy in aging.

[The age-related characteristics of autophagocytosis in different tissues of laboratory animals]. / Vozrastnye osobennosti autofagotsitoza v razlichnykh tkaniakh laboratornykh zhivotnykh.

To elucidate the role of autophagy in age-related lipofuscinogenesis, the accumulation of autophagic vacuoles (AV) caused by the blockade of their fusion with lysosomes under the effect of vinblastine was studied in cells of various tissues (hepatocytes, pinealocytes, epitheliocytes of choroid plexus, cardiomyocytes, neurons) in mice and rats of different ages. The degree of AV accumulation in cells of old animals did not exceed that in adult ones. The results suggest that the accumulation of lipofuscin (LF) in aging cells is mainly due to the disturbance of lysosomal degradation or removal of LF out of cells rather than to the increase in autophagy.

Ultrastructural study of clinically uninvolved skin of patients with pemphigus vulgaris.

We investigated skin biopsies from pemphigus vulgaris (PV) patients by light, fluorescent and electron microscopy in order to study the ultrastructural appearances of epidermis at the pre-acantholytic stage. The biopsies were obtained from uninvolved forearm skin in 10 patients with PV in the acute stage of the disease, from perilesional skin of the same patients as well as from the forearm skin of 10 healthy subjects. Light microscopy showed no pathological changes in clinically uninvolved skin of pemphigus patients. Direct immunofluorescence confirmed the presence of IgG auto-antibodies fixed in intercellular space of the spinous-cell layer of uninvolved skin. Electron microscopy of the uninvolved skin biopsies revealed the following changes: disintegration of desmosomes of spinous cells with their replacement by finger-shaped protrusions of cytoplasm; clarification of the nuclear matrix; widening of the perinuclear slit; an increased number of secondary lysosomes in cells; oedema and swelling of mitochondria with destruction of their cristae. The cells retained their polygonal shape and the intercellular distance did not increase. We conclude that at the pre-acantholytic stage the breakage and dissolution of desmosomes precedes the increase in the intercellular space.

[The nature of and the interrelations between the concepts of "life","disease" and "aging"]. / Sushchnost' i vzaimosviaz' poniatii "zhizn'", "bolezn'" i "starenie".

An attempt is made to consider disease and aging following from the concepts of the essence of life. The proposed definition of life represents a modified Engels' (1878) definition. Proceeding from the analysis of possible mechanisms of different disturbances in the life process leading to a decreased probability of the organism existence it is concluded that disease develops either as a result of hereditary changes in the genome or due-to disorders in its realization under certain unfavorable conditions. Aging is determined by the properties of the genome itself and develops in connection with age increase.

[The ultrastructural changes in the clinically unaffected skin of patients with pemphigus vulgaris]. / Ul'trastrukturnye izmeneniia klinicheski na porazhennoi kozhi bol'nykh vul'garnoi puzyrchatkoi.

Analysis of the results of light optic, immunofluorescent, and electron microscopic studies of biopsy specimens of intact skin of forearms and of the skin adjacent to lesions in 10 patients with untreated pemphigus vulgaris has lead the authors to a conclusion on the uniform pattern of ultrastructural changes in all sites of the epidermis in pemphigus patients. A combination of the signs of involvement of prickle and basal-cell structures (desmosomes and mitochondria) and of signs of cellular metabolic activation (euchromatism, active status of the nucleolus) anticipate the development of intra- and extracellular changes in pemphigus.

[Ultrastructural manifestations of blood-tissue barrier reactions to acute hypoxia in old animals]. / Ul'trastrukturnye proiavleniia reaktsii gistogematicheskikh bar'erov starykh zhivotnykh na ostruiu gipoksiiu.

Readjustments occurring in different blood-tissue barriers (the brain, endocrine glands, heart, liver, jejunum) in response to acute hypoxia in old animals develop against the background of the preceding age-specific changes, including the adaptive ones. This results, first, in a significant narrowing of the adaptive reaction range and, second, acceleration and deepening of destructive processes in tissues and prolongation of the restoration period.