老化红细胞肌醇磷脂代谢的探讨

本文用密度梯度离心法,将红细胞分成年轻及老化两部分,分别测定了膜磷脂、甘油二酯及磷酸肌醇的含量,蛋白激酶C在膜内及胞浆内的分布及其对膜蛋白的磷酸化。实验结果表明老化及年轻红细胞磷脂含量,除磷脂酰胆碱明显降低外,其他均无差异。老化红细胞甘油二酯含量低于(P<0.01)年轻者,而磷酸肌醇两者含量相似。老化红细胞膜内蛋白激酶C活性远比年轻者高,而在胞浆内活性则相反。说明老化红细胞膜蛋白激酶C活性增高,可使红细胞膜蛋白4.1、4.2、4.5和4.9进行磷酸化,这些膜蛋白与维持红细胞形态及功能有关,可能它们的磷酸化是老化红细胞变形性降低,脆性增加的因素之一。     

Characterization of "fetal-type" acetylcholinesterase in hemin-treated K562 cell culture

The alteration of acetylcholinesterase (ACHE) activity, a marker enzyme of erythroid differentiation, was studied during the hemin-induced erythroid differentiation of K562 human leukemia cells in suspension culture. The kinetics of postinduction differentiation was followed by determining the hemoglobin (Hb) content and the ACHE activity of cells. Embryonic hemoglobins as well as small quantities of fetal Hb (HbF) were synthetized by stimulated cells. The peaks of ACHE activity preceded the highest level of Hb content and, following induction, reached their pinnacles at 72 and 120 hours, respectively. These data indicate that ACHE activity is an earlier and more sensitive marker for hemin-induced erythroid differentiation of K562 cells than is elevated Hb content. Electrophoretic mobility of ACHE from hemin-treated cells proved to be the fetal type, but after incubation with neuraminidase, the rate of migration decreased to the level of the adult type enzyme.     

Acetylcholinesterase in aging of human erythrocytes]

Acetylcholinesterase possessed a different activity in the membranes of young, mature and old human erythrocytes - it was greatest in the mature and least in the old cells. In the young and mature erythrocytes the enzyme existed in the form of 3- and in the old ones in the form of 2-molecular components. The data obtained suggested that the changes in the structural organization of acetylcholinesterase in the red cell membrane had a direct relationship to the aging of these cells.     

Localization of acetylcholinesterase in granule-containing cells of glomus-like bodies in pre- and postnatal rabbits by electron microscopy

The distribution of acetylcholinesterase (ACHE) was studied in the granule-containing cells which constitute the glomus-like bodies found near the origin of the great vessels in pre- and postnatal rabbits. Karnovsky's method for localization of ACHE at the electron-microscope level was used and suitable controls were carried out. In the granule-containing cells, ACHE reaction product was evident in the perinuclear cisternae and cisternae of the rough endoplasmic reticulum as well as at the cell membrane. ACHE activity was also localized at the axolemma of unmyelinated axons found near the granule-containing cells and around afferent synaptic terminals to these cells. Possible functions of ACHE associated with the monoamine-storing granule-containing cells are presented.     

Red cell ageing: phagocytosis and life-span of young and old erythrocytes fractionated by centrifugation

Young and old red blood cells, separated by centrifugation on the basis of differences in cell density, were submitted to phagocytosis by either autologous human alveolar macrophages or syngeneic murine bone-marrow macrophages. Young cells adhere to macrophages, but to a much smaller extent than old ones. The influence of both type and quality of the separation procedure on the differences observed between the two erythrocyte subpopulations is discussed in the light of the half-life times of murine young and old red blood cells. Fractionation according to age was obtained following the method of Murphy (1973) and glutamate oxalo-acetate transaminase activity was measured and used as an indicator of both cell age and separation.     

Total sialic acid content of glycophorins during senescence of human red blood cells.

Glycophorins extracted from membranes of young and old human red blood cells have within an error of +/- 1.5% the same sialic acid content when referred to a relative measure of the number of glycophorins. The degree of surface iodination in glycophorins, which was shown to be the same in young and old cells, served as this relative measure. This finding implies that senescent human red blood cells hardly reveal desialylated surface proteins (less than or equal to 3%). However, the sialic acid content per cell was repeatedly reported to be 10 to 15% lower in old than in young cells. Therefore, we conclude 1) that human red blood cells lose intact glycophorin together with membrane during red blood cell senescence, and 2) that removal of desialylated and senescent red blood cells from the circulation proceeds by different routes.     

In vivo survival of selected murine carrier red blood cells after separation by density gradients or aqueous polymer two-phase systems.

In order to explore possibilities of using erythrocytes as carrier systems for delivery of pharmacological agents, we have studied the in vivo survival of murine carrier red blood cell populations enriched in young or old cells. Hypotonic-isotonic dialysis has been used to modify the cells as carrier systems and Percoll/albumin density gradients or counter-current distribution in aqueous polymer two-phase systems to separate them according to age. Hypotonic-isotonic dialysis produces a decrease in the red blood cell populations in vivo survival rate (from 9.5 to 7.8 days). Among the cells modified as carriers, the enriched young red blood cell populations show a higher in vivo survival (half-life 6.5-7.4 days) than populations made up of predominantly old red blood cells (half-life 4.7-6.2 days). Half-life of young or old circulating red blood cells was approximately one day longer when these cells were separated by counter-current distribution rather than by Percoll density gradients. Based on these results, hypotonic-isotonic dialysis of whole and enriched young or old red blood cell populations, with higher or lower survival rates, can be considered as a useful tool for modification of these cells as carriers. The final outcome of such changes can be translated into better control of plasma drug delivery during therapy.     

Ouabain Binding and Potassium Transport in Young and Old Populations of Human Red Cells

Human red blood cells were separated according to density by centrifugation through mixtures of phthalate esters. The densest 20% of the erythrocyte population (old cells) had reduced volume and water content compared to the lightest 20% of the cells (young cells). Corpuscular hemoglobin content was unchanged. Young cells had 50% more potassium (K⁺) than old cells, but their total intracellular concentration was only slightly higher; old cells had a small increase in sodium (Na⁺) concentration. Active K⁺ transport of young cells was 37% higher than that of old cells. [³H] + Ouabain binding revealed that this difference was the result of more K⁺ pump sites on young cells, which bound 530 ouabain molecules per cell at 100% K⁺ pump inhibition, as compared to 400 for old cells; unseparated cells bound 450-500 molecules. The relative rates of ouabain binding were identical for the two cell types. Old cells exhibited a greater passive permeability to K⁺, haying a rate coefficient for ouabain-insensitive K⁺ influx 1.8 times that of young cells. There is evidence to suggest that in the face of reduced pump activity this augmented K⁺ “leak” might enhance the osmotic stability of the old cells and function to lengthen their life span.     

Retracted: Upregulation of acetylcholinesterase caused by downregulation of microRNA-132 is responsible for the development of dementia after ischemic stroke

MicroRNA-132 (miR-132) has been shown to participate in many diseases. This study aimed to understand the correlation between the level of miR-132 and the severity of dementia post-ischemic stroke. An online tool ( www.mirdb.org ) was used to find the miR-132 binding site in acetylcholinesterase (ACHE) 3′-untranslated region (UTR), followed by a luciferase reporter assay to validate ACHE as a miR-132 target. A similar relationship between miR-132 and ACHE was also established in cerebrospinal fluid samples collected from human subjects. A negative correlation was established between ACHE and miR-132 by measuring the relative luciferase activity. Meanwhile, Western blot analysis and real-time polymerase chain reaction were also conducted to compare the levels of ACHE messenger RNA and protein between two groups (dementia positive, n = 26 and dementia negative, n = 26) or among cells treated with miR-132 mimics, ACHE small interfering RNA, and miR-132 inhibitors. As shown in the results, miR-132 can reduce the expression of ACHE. Further experiments were also carried out to study the effect of miR-132 and ACHE on cell viability and apoptosis, and the results demonstrated that miR-132 enhanced cell viability while suppressing apoptosis. In addition, ACHE reduced cell viability while promoting apoptosis. miR-132 targeted ACHE and suppressed its expression. Additionally, miR-132 and ACHE have been shown to affect the cell viability and apoptosis in the central nervous system.     

Altered enzyme function and premature sequestration of erythrocytes in aged individuals

Experimentation performed to determine the parameters of the life-span of the erythrocyte in hosts of various ages have determined that, in aged individuals, the rate of turnover of cells is considerably increased over that observed in young individuals. These observations are based on studies in humans, rats, mice, and rabbits in which either in situ 59Fe labelling or age-density gradient separation were used. The mechanisms for the recognition of the effete red cell in the aged host and the nature of the membrane alterations that bring about the premature sequestration are not fully understood. However, it has been consistently observed that the red cells of aged individuals have higher levels of IgG bound to their membranes than do young cells, with the most dense cells having the highest levels of immunoglobulin. Studies of most enzymes, particularly those involved in protection against oxidative damage have shown reduced activity as a function of both cell and donor age. Evidence of enzyme damage has been observed even in the youngest circulating red blood cells of old individuals. This fact leads us to hypothesize that the erythrocyte of the aged individual as it differentiates and is released from the bone marrow is less functional and partially damaged. The erythrocytes of both old and young individuals age in the circulation, accumulating subtle alterations that are recognized by the immune and/or reticuloendothelial systems and lead to sequestration. The cells of the elderly individual accumulate a greater degree of damage due to their initially reduced capacity to protect themselves from environmental stress. These alterations eventually bring them to their early sequestration.     

Alteration of alpha-spectrin ubiquitination due to age-dependent changes in the erythrocyte membrane.

Mammalian red blood cell alpha-spectrin is ubiquitinated in vitro and in vivo [Corsi, D., Galluzzi, L., Crinelli, R., Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935]. This process shows a cell age-dependent decrease, with senescent red blood cells having approximately one third of the amount of ubiquitinated alpha-spectrin found in young cells. In-vitro ubiquitination of alpha-spectrin was dependent on the source of the red cell membranes (those from older cells are less susceptible to ubiquitination than those from younger cells), on the source of ubiquitin-conjugating enzymes (those from older cells catalyze the process at a reduced rate compared to those from younger cells) and on the ubiquitin isopeptidase activity (which decreases during red cell ageing). However, once alpha-spectrin has been extracted from the membranes of young or old red blood cells, it is susceptible to ubiquitination to a similar extent regardless of source. This suggests that it is the membrane architecture, and not spectrin itself, that is responsible for the age-dependent decline in ubiquitination. Furthermore, spectrin oligomers, tetramers and dimers are also equally susceptible to ubiquitination. As spectrin ubiquitination occurs on domains alphaIII and alphaV of alpha-spectrin, and domain alphaV contains the nucleation site for the association of the alpha- and beta-spectrin chains, alterations in ubiquitination during red cell ageing could affect the stability and deformability of the erythrocyte membrane.     

Acetylcholinesterase activity in chronic renal failure

Twenty healthy subjects and 39 Chronic Renal Failure patients (CRF-patients) maintained on chronic hemodialysis were used in this investigation to study the changes in acetylcholinesterase (AChE) activity of red blood cells (RBCs). The CRF-patients were all undergoing hemodialysis treatment. AChE activity from the CRF-patients was determined before and after dialysis. An additional objective was to study the effect of chronic renal failure on human red blood cell aging. Blood samples were drawn from controls and CRF-patients in tubes containing EDTA or sodium heparin as an anticoagulant. Red blood cells were purified to avoid interference with monocytes, reticulocytes and leukocytes. The purified RBCs were subfractionated into young (y) (1.08-1.09), mid (m) (1.09-1.11) and old (o) (1.11-1.12) percoll density (g/mL) fractions using a discontinous percoll gradient. The mean +/- SD AChE per gram hemoglobin (U/g Hgb) activities in whole blood (WB), purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) in CRF-patients were 31.2+/-3.43, 29.3+/-3.26, 30.4+/-3.91, 25.1+/-5.25, 17.1+/-6.02 in females and 29.8+/-5.39, 28.8+/-5.29, 28.7+/-5.29, 23.7+/-5.39 and 16.0+/-5.60 in males. AChE activity from CRF-patients were higher than that found in the control subjects. The aging of human RBCs in both the controls and CRF-patients showed a progressive reduction in AChE activity. AChE activity of RBCs from female CRF-patients were significantly higher (p < 0.05) than that of the female control subjects. The RBCs isolated from male CRF-patients showed a higher AChE activity than control males, but a significant difference was only observed with the mid-age-cells. These studies further indicate that AChE activity remained insignificantly different in the various density based age subfractions of RBCs of both CRF-patients and controls.     

A new cell surface marker of aging in human diploid fibroblasts

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continuously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity di not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.     

Effects of marrow donor and recipient age on immune responses

This report describes treatments to restore diminished splenic immune responses of old mice. Lethal irradiation, followed by young bone marrow and infant thymus transplants, restored the T cell mitogen response and the antibody-forming cell response against sheep red blood cells in the old mice. Although old bone marrow cells restore these immune responses in young recipients, as well as do young bone marrow cells, old bone marrow in old recipients did not improve their levels of response. Longevities of old recipients with rejuvenated responses were not increased, and aging of tail tendon collagen was not affected. The effect of lethal irradiation before the marrow transplant was shown to be minimal, by the use of unirradiated old W-anemic recipients. Parabiosing young mice with old partners caused impairment of these two immune responses in the young partners without enhancing them in the old partners. The old partners did not have increased longevities. To explain these results, we suggest the following hypothesis: old bone marrow contains precursors that produce suppressive factors or cells when in an old environment but not when in a young environment. However, these factors, if allowed to develop in an old environment, can function in a young parabiosed partner.     

Participation of the adenylate cyclase system in inductive synthesis of acetylcholinesterase in the brain]

The enhancement of the brain acetyl cholinesterase (ACHE) activity in rats by the intraventricular injection of adrenaline or dibutiryl analogue cyclic adenosine-3',5'-monophosphate (cAMP) was shown to be due to the enzyme inductive synthesis. ACHE induction manifests itself more in the subcortical white matter than in the cortex. The stimulating effect of adrenaline on the ACHE activity is suppressed under the beta-adrenoreceptor block, while the cAMP effect remains unchanged. On the contrary, the block of the alpha-adrenoreceptors stimulates the enzyme synthesis induction. The effects of adrenaline and cAMP are of the same direction and are realized through the beta-adrenoreceptors. The enhancement of ACHE activity during the block of the alpha-adrenoreceptors is accounted for by the elimination of their inhibitory influence on the beta-adrenoreceptors.     

Vitamin E in young and old human red blood cells

Young and old human red blood cells contain about the same amount of alpha-tocopherol, a compound which has previously been shown to be the major lipid-soluble, chain-breaking antioxidant present in such cells. Since red blood cells lose up to ca. 20% of lipid material from their membrane as they age, the alpha-tocopherol/membrane-lipid ratio actually rises with age rather than declining as might have been expected on the basis of the free radical theory of aging. The alpha-tocopherol/arachidonic acid moiety ratios increase in the order: young red blood cells less than old red blood cells less than plasma, which argues against the suggested membrane stabilizing effect of alpha-tocopherol/arachidonic acid moiety complexes.     

Studies on NADH-cytochrome b5 reductase activities in hemolysates of human and rabbit red cells by isoelectric focusing

NADH-cytochrome $\text{b}{}_5$ reductase activities in hemolysates of young and old human erythrocytes, and in hemolysates of rabbit reticulocytes and erythrocytes were measured after the separation of the enzyme from the bulk of hemoglobin only by isoelectric focusing. In any cases, a single main peak of the enzyme activity was detected after the electrophoresis in the fraction with pH 6.8 and 8.3 for human and rabbit red cells, respectively. The rabbit enzyme showed more than 30 times higher enzyme activity than that of human erythrocytes under the standard assay conditions. Significant differences of Micahelis constants for cytochrome $\text{b}{}_5$ of the enzyme were found between young and old human erythrocytes, and also between human and rabbit red cells.     

The characteristics of cholinesterase distribution in the development of the human and mammalian brains]

Histochemical studies have been made on the distribution of acetyl- and butyrylcholinesterases (ACHE and BCHE) in various parts of the human and rat brain. Statistical analysis showed that at the 8th week, the highest ACHE activity in the human foetus is observed in the intermediate and plexiform layers of the cerebral cortex. The highest BCHE activity was found in the ependymal layer of various cerebral regions. High BCHE and ACHE activities were noted in the dorsal thalamus and epithalamus. In 10-week human foetuses, total high level of ACHE and BCHE was revealed in various nuclei of the thalamus and subcortical structures of the forebrain (Meynert nucleus, nucleus caudatum). In rats, the highest ACHE activity at the 14th day of prenatal life was found only in subcortical structures of the forebrain. Accumulation of BCHE activity in some of the thalamic nuclei of rats begins at the 10-17th day of postnatal life.     

Cell lines from old immunodeficient donors give normal responses in young recipients.

Two different immune responses were compared in spleen cells obtained from old and young CBA/HT6J mice. Spleen cells from old mice (23 to 33 months) responded about half as well as did spleen cells from young mice (4 to 10 months) in the adoptive transfer anti-sheep red blood cell (SRBC) plague-forming assay, and caused slightly less than half the uptake of tritiated thymidine in response to phytohemagglutinin (PHA) in vitro. Marrow stem cell from some of the old and young mice whose splenic immune responses were tested were transplanted into irradiated young CBA/CaJ recipients. Seven to 17 weeks later these same immune responses were tested in the spleen cells of these young recipients, and the T6 chromosome marker was used to identify donor cells. Old animals' responses varied greatly, perhaps due to suppressing cells or factors in some individuals. Therefore, cells were never pooled and the responses of receipients were compared to the responses of the donor whose marrow had populated them. The response for a particular old donor, or for the recipients of its stem cells, was divided by the response for the young control used with that donor, or for its stem cell recipients. This was called the old/young ratio. With original donors with an old/young ratio for the SRBC response of (mean +/- S.D.) 0.35 +/- 0.14, The old/young ratio for that same response in the recipients was significantly improved to 1.26 +/- 0.71. In original donors with an old/young ratio for the PHA response of 0.44 +/- 0.17, the old/young ratio in the recipients improved significantly to 0.86 +/- 0.27. Thus, little or none of the decline with age in these immune responses was intrinsic to the old lymphoid stem cells.     

Copper activation of superoxide dismutase in rat erythrocytes

Several lines of evidence suggested that copper can activate a preexisting pool of superoxide dismutase (SOD) apoprotein in erythrocytes from copper-deficient rats. First, feeding adequate copper to copper-deficient rats raised initially low erythrocyte SOD activities to normal values in under one-third the time needed to replace the entire red cell population. Moreover, copper injection (1 mg Cu/kg, sc) doubled erythrocyte SOD activity levels in 16 h. Since protein synthesis is restricted in mature erythrocytes, these results imply that copper activated apoSOD in vivo. Furthermore, injected copper raised SOD activity contents of both young and old erythrocytes. Neither dietary copper status nor copper injection influenced red cell SOD immunoreactive protein levels. In contrast, copper injection increased the amount of copper associated with the SOD activity peak region resulting from gel filtration of hemoglobin-free erythrocyte proteins on Sephadex G-75. Copper ions (3 microM) elevated SOD activity levels in vitro by 63% in 4 h in intact red cells from copper-deficient rats. No activation took place in lysed red cells from the same rats or in intact cells from copper-adequate rats. These results all suggest that copper can activate SOD apoprotein in erythrocytes by a specific, saturable process.