Age-dependent alterations of DNA synthesis. Terminal deoxynucleotidyl transferase and DNA polymerase activities in bone marrow subpopulations from mice

The decrease of functional capacity of cellular immunity during ageing seems to be due to cellular changes of stem cells, particularly in the growth properties and the cell density in T-cell subsets. We approached this problem at the molecular biological level by quantifying the key enzymes necessary for DNA synthesis in bone marrow cells from mice: deoxynucleotidyl transferase (TdT) and DNA polymerase α. The bone marrow cells were fractionated on a discontinuous bovine serum albumin density gradient and the extractable enzyme activities (expressed per 10⁸ nucleated cells in the respective fraction) were determined.TdT activity was found to decrease markedly during ageing. Mature animals contain only 34% and senescent animals only 13% of the activity observed in immature mice. From the density distribution analysis it was found that a shift of TdT-containing cells to the lower density occurs.The specific DNA polymerase α activity also decreases in bone marrow cells with age. While the overall activity amounts in immature cells to 78 enzyme units/10⁸ cells, it decreases in mature cells to 57 units/10⁸ cells, and in cells from senescent animals to 36 units/10⁸ cells. Density distribution analysis of the cells shows that the highest activity is observed in the low-density fraction. From these experimental data we conclude that in the fractions containing precursor T-cells, a reduced number of proliferating cells is present.     

Healing of skin incision wounds treated with topically applied BAPN free base in the rat

β-Aminopropionitrile as free base (BAPN) was applied onto the incised or intact skin of rats at the dose of 5, 20, 100, and 200 μl for 9 days, twice daily. Breaking strength of the skin wound or intact skin was significantly reduced at doses of 20 μl and higher; body weight growth was significantly retarded at the two highest dosages. It is concluded that at a given dose (20 μl) collagen polymerization (evaluated by reduced breaking strength and increased extractability of collagen) was specifically inhibited by BAPN. Furthermore, no evidence of topical or general toxic effects were observed, as reflected in histology, body weight growth, and behavior of the rats. Acute LD₅₀ of BAPN base and fumarate, administered either ip or topically, was determined in mice. While BAPN base in ip administration shows LD₅₀ of 1.15 g/kg, in cutaneous application it is more than 12.8 g/kg. It is suggested that topically applied BAPN base is percutaneously absorbed and affects collagen polymerization in the skin and adjacent tissues.     

Age-related alterations in cultured human fibroblast membrane structure and function

Membrane enzyme activities, lipid composition, and fluorescence probe characteristics in isolated plasma membranes, microsomes and mitochondria of cultured human fibroblasts were used to determine if structural alterations occurred as a function of donor age. The cells were sex matched and allowed to undergo approximately 8 population doublings under identical culture conditions. Plasma membrane (Na⁺,K⁺)-ATPase, microsomal NADPH cytochrome c reductase, and mitochondrial succinate cytochrome c activities showed variation as a function of increasing donor age but these changes were not statistically significant. At the same time the cholesterol/phospholipid molar ratio was unaltered in plasma membranes, decreased 50% in microsomes, and unchanged in mitochondria with increasing donor age. The phosphatidylcholine/phosphatidylethanolamine ratio increased in all three membrane fractions with increasing age of the fibroblast donor. The ratio of unsaturated/saturated fatty acids decreased in the phospholipids of microsomes but not of plasma membranes or mitochondria. The structural properties of the membranes were determined with two different fluorescence probe molecules, trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene. These probe molecules indicated that the fluorescence lifetime and/or fluorescence polarization of the trans-parinaric acid probe decreased in microsomes, mitochondria, and in the plasma membrane, such that the limiting anisotropy, indicative of restrictions to probe motions, was significantly lower (high fluidity) with increasing subject age in plasma membranes, microsomes and mitochondria. The trans-parinaric acid fluorescence lifetime displayed two components in plasma membranes, microsomes, and mitochondria, a finding consistent with the co-existence of fluid and solid membrane lipid areas in the cultured human fibroblast subcellular membranes. The trans-parinaric acid partitioned preferentially into solid membrane     

Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase)

Microtubules have been isolated from immature (3-4 weeks' old) and old (11-13 years' old) bovine brains. Quantitative studies revealed that the concentration of extractable microtubule protein per gram of wet brain decreased from 0.47 mg (immature animals) to 0.34 mg (old animals). The major components of microtubule protein (tubulin and high-molecular-weight microtubule-associated proteins) do not undergo an age-correlated change. Determination of the endogenous protein kinase activity revealed that the activity associated with "immature" calf brain microtubules was six times higher than the activity present in "old" preparations. In contrast, the stimulatory effect of cyclic AMP on protein phosphorylation in microtubules from old bovine brains exceeds nine-fold the value obtained from immature animals. After addition of casein (exogenous acceptor), the basal activities increased in both preparations without altering the age-correlated difference in the specific activity. By comparing the radioactivity pattern of sodium dodecyl sulfate polyacrylamide gels after autophosphorylation of microtubule protein with [gamma-32P]ATP, 1.5 moles of phosphate per mole of high-molecular-weight microtubule-associated protein were estimated to be incorporated in preparations from immature animals and 0.9 mole of phosphate per mole of associated protein in the experiments with "old" microtubule protein. Adenosine triphosphatase activity, associated with the high-molecular-weight microtubule-associated protein 1, was determined to be 15% reduced in preparations from old animals, compared to the activity in "young" preparations. In contrast, the guanosine triphosphatase activity increased five-fold during ageing; the higher activity of this enzyme was observed both during the initial and the steady-state phases of microtubule formation.     

A cell-determined deficiency in the processing of gag proteins of murine leukemia virus 334C

A murine leukemia virus was cloned by a method which yielded cell clones each chronically infected with a virus clone. One of these produced virus which had an abnormal protein pattern characterized by increased amounts of certain proteins normally present in trace amounts. Immunoreplica analysis showed that these proteins were related to the major capsid protein p30 and suggested that they were uncleaved precursor proteins or other high-molecular-weight proteins derived from the gag gene. The major species of RNA in this clone were the same size as those in a clone producing normal virus. When uninfected cells were infected with virus produced by this clone, the progeny had a normal protein pattern. Finally, this clone was abnormal in its growth characteristics in that the cells died rapidly upon becoming confluent. This cell clone, therefore, seems to have a cell-determined deficiency in the processing of leukemia virus proteins. Electron microscopy showed that this cell line produced an abnormally high proportion of virus particles which had an immature morphology.     

Na+ and K+ concentrations and the regulation of protein synthesis in Sindbis virus-infected chick cells.

Selective inhibition of host-specified protein synthesis in Sindbis virus-infected chick cells occurs at the level of the initiation of translation of host cell mRNAs. This inhibition is temporally correlated with an increase in the intracellular Na⁺ concentration and a decrease in the intracellular K⁺ concentration. Raising or lowering the NaCl concentration of the medium in which uninfected chick cells are incubated also inhibits protein synthesis, interferes with the initiation of translation of cellular mRNAs, and changes the intracellular monovalent cation concentrations. Lowering the NaCl concentration of the medium decreases intracellular K⁺, while raising the extracellular NaCl concentration increases both intracellular Na⁺ and K⁺. The selective inhibition of host cell protein synthesis, both by Sindbis virus and by altered NaCl media, appears to result from changes in the intracellular na⁺ and K⁺ concentrations. Under conditions of decreased K⁺ and/or increased Na⁺, viral and a few host mRNAs (such as that for chick actin), which apparently possess higher affinity for some component(s) of the initiation complex (high affinity mRNAs), can initiate while the majority of cellular mRNAs (low affinity mRNAs) cannot.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.     

Differential effects of ouabain on host- and sindbis virus-specified protein synthesis.

Ouabain, a specific inhibitor of the membrane-associated Na⁺/K⁺-ATPase (sodium pump), has a differential effect on protein synthesis in Sindbis virus-infected and uninfected chick embryo fibroblast (CEF) cultures. At low concentrations, cell-specified protein synthesis is inhibited while Sindbis virus-directed protein synthesis is stimulated. At higher ouabain concentrations, which inhibit protein synthesis in uninfected CEF cells 95% or more, viral protein synthesis continues. Ouabain treatment of uninfected CEF cells raises the intracellular Na⁺ concentration and lowers the intracellular K⁺ concentration. The effect of Sindbis virus infection is similar. The changes in intracellular Na⁺ and K⁺ concentrations appear responsible for the selective inhibition of the translation of host cell mRNAs in Sindbis virus-infected or ouabain-treated CEF.     

Impaired glycolysis of human lymphocytes during aging

When peripheral lymphocytes from young persons are stimulated in vitro with phytohemagglutinin, an increase in the levels of all the glycolytic enzymes occurs concomitantly with blastogenesis. The specific activities of enzymes increase approximately 200%, with a greater induction of the latter half of the pathway. The increases do not represent a general enzyme induction, since nonglycolytic enzymes such as glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase do not increase during transformation.Human lymphocytes from a geriatric population were also subjected to mitogen stimulation under identical conditions. The initial levels of the enzymes were essentially identical in lymphocytes from young and old subjects as were cultured controls which received no mitogen. However, during mitogen stimulation the cells from the old subjects failed to incrase the glycolytic enzymes. This inability to activate glycolysis may be related to the decline in cell-mediated immunity which is known to occur with advacing age.     

Neurotransmitter enzymes in telencephalon,brain stem and cerebellum during the entire life span of the mouse

Neurochemical analysis of neuronal function was undertaken by measuring the activities of cholinacetyltransferase (CAT), acetylcholinesterase (AChE), and glutamic acid decarboxylase (GAD), in the telencephalon, brain stem and cerebellum of the mouse.Cholinergic activity was first expressed in the 10-day embryonic brain stem, which showed a relatively high CAT activity at birth. Postnatal brain stem development was characterized by a rapid and parallel increase in CAT and AChE. Although AChE peaked at 1 month, CAT activity was not achieved until 1 year. Acetylcholine synthesis was initiated in the 12-day embryonic telencephalon and a steady age-related increase in CAT was maintained until birth. A lag in both CAT and AChE activities was recorded during the first week of postnatal telencephalon development. Cerebellar CAT was low at birth, and increased irregularly to reach a maximum by 1 month. In contrast, postnatal cerebellar AChE activity increased steadily over the same time period.The GABA-ergic neuronal system matured rapidly in each brain region, and was unaffected by aging.Although the brain stem precociously expressed cholinergic activity, it was the region most susceptible to deterioration during aging.Telencephalon CAT activity was unaffected by aging and in the cerebellum, a significantly reduced level of CAT was only found in truly senescent animals.The decreased cholinergic function during senescence was not due to either increased proteolysis or to alteration in the molecular form of the cholinergic enzymes.     

Life-lengthening effects of γ-radiation on the adult housefly, Musca domestica

Effects of γ-irradiation on the life span of the adult housefly (Musca domestica) were examined in order to elucidate the relationship between metabolic rate, irradiation and longevity. Adult houseflies of both sexes, exposed to 0, 20, 40 and 66 kR of γ-radiation, were housed under conditions of relatively high or low physical activity. The mean longevity of high activity males exposed to 20 and 40 kR exhibited a significant increase, whereas the average life span of all female populations, low activity males, and high activity males exposed to 66 kR was significantly decreased following irradiation. Radiation exposure caused a reduction in the rate of oxygen consumption in both sexes as well as a decrease in the incidence of wing loss in the male flies. Analysis of the results suggests that radiation-induced life-lengthening in the housefly is a consequence of reduced metabolic activity.     

Cardiac morphologic alterations in acute minoxidil cardiotoxicity in miniature swine

Minoxidil, a vasodilating antihypertensive drug, was given orally at 10 mg/kg daily for 2 days to twelve 25- to 35-kg miniature pigs. Twelve control pigs were also studied. Minoxidil-treated pigs had tachycardia and hypotension and were killed 24 hr after the second dose. Gross examination showed diffuse hemorrhage in left atrial epicardium in all pigs, and also in ventricular epicardium (2 of 12 pigs) and endocardium (3 of 12 pigs). Pale areas of necrosis were observed on incision of the left ventricular papillary muscles in 3 pigs. Light and electron microscopic studies showed acute vascular damage with hemorrhage in the left atrial epicardium. Affected arterioles had endothelial cell swelling and transmural and perivascular accumulations of leukocytes, edema fluid, fibrin clumps, and erythrocytes. The swollen endothelial cells had large, irregularly shaped nuclei with abundant euchromatin; mitotic figures were frequent. The cytoplasm contained numerous polysomes and cisterns of rough endoplasmic reticulum. Fibroblasts adjacent to damaged vessels had edematous cytoplasm and increased amounts of rough endoplasmic reticulum. In the affected left ventricular papillary muscles, necrotic myocytes showed contraction bands, mitochondrial matrical densities, lipid accumulation, initial lysis of I bands, and pyknotic nuclei. The lesions were judged to result from two mechanisms: (1) hemorrhagic lesions from drug-induced vascular injury centered on epicardial and subepicardial arterioles and (2) papillary muscle necrosis from ischemic injury from hypoperfusion during minoxidil-induced tachycardia and hypotension.     

Growth,structure and function of baboon aortic smooth muscle cells in culture

Procedures for the successful culture of aortic medial smooth muscle cells (SMC) from the baboon (Papio cynocephalus) have been described. Growth rates, plating efficiency, and doubling times were determined. SMC showed increasing growth with increasing concentrations of heat-inactivated homologous serum. Growth on glass was accelerated relative to plastic surfaces, a difference reflected in the more numerous and extensive attachment sites on glass and more extensive cellular spreading. Attachment sites on both glass and plastic were associated with short parallel arrays of thin filaments (50 Å). The fine structure of SMC grown on plastic or glass was examined at intervals from 7 to 74 days in culture. Cells exhibited many of the features traditionally associated with cultured SMC, including basement membrane material and extracellular matrix, predominantly thin cytoplasmic filaments (40–80 Å) with dense bodies, coated pits and vesicles, caveolae and pinocytotic vesicles, and both cell-cell and internalized gap junctions. Changes with increasing time in culture included an increased extracellular matrix, more numerous lipid inclusions, residual bodies, myelin forms, multivesicular bodies, and crystalline lysosomal inclusions. Membranous multivesicular blebs and extruded autophagosomes were also seen with increasing frequency. An increasing culture age was also associated with changes in organelles, including less prominent Golgi profiles, more free ribosomes, dilatation of the cisternae of the RER, and less prominent shorter profiles of mitochondria with increasing electron density. Cultured SMC exhibited both a continuous and a homogeneous ruthenium red-staining glycocalyx around their perimeter, and the ability to respond to the polycationic ligand, CF, with a redistribution of surface-associated anionic binding sites. Additionally, they have been shown to possess high-affinity LDL receptors (K_d = 1 × 10^?7M) with binding showing half-maximal saturation at 20 μg/ml.     

Age-related changes in immune function of rats and the effect of long-term hypophysectomy.

The effect of age on the ability of rats to raise antibodies to sheep erythrocytes and on the number of IgM plaque-forming cells in the spleen was investigated. An age-related decline in both parameters was observed. Additionally, the possibility that long-term hypophysectomy coupled with minimal replacement therapy might result in a delay and/or reversal of the age-related decline in immune function was studied. It was observed that long-term hypophysectomized rats responded better to immunization with sheep erythrocytes than did their age-matched unoperated littermates. The possible relationship of this to aging is discussed.     

Age-associated changes in poly(adenylate) polymerase of rat liver and brain

The activity of poly(adenylate) polymerase was measured in nuclear lysates of rat brain and liver of animals ranging from 25 to 300 days. The activity was 26 nmoles AMP incorporated per h per mg of protein in liver and 8 moles per h mg in brain for 25-day-old rats, and declined to 1.0 and 3.3 for liver and brain, respectively, in 300-day-old rats. At all ages the liver showed a linear time course and had a Km for ATP of 0.5 mM. The brain enzyme gave a non-linear time course at all ages with an ATP optimum of 0.2 mM. Mixing enzyme preparations from young and old animals produced additive results. Mixing brain and liver enzyme produced greater than additive results. No substantial increase in activity was observed when enzyme was prepared from old or young partially hepatectomized rats as compared to sham-operated controls.     

GABA receptors distribution in rat substantia nigra

The perikarya of the dopaminergic and non-dopaminergic neurons contained in the substantia nigra were selectively destroyed by a proper, local injection of 6-hydroxydopamine or kainic acid, respectively. Both lesions resulted in a marked decrease of the nigral GABA-binding sites. The effect of 6-hydroxydopamine was restricted to the high affinity receptors, while kainic acid specifically decreased the low affinity ones.     

Biochemical and functional properties of large and small dense-core vesicles in sympathetic nerves of rat and ox vas deferens

A comparative study of the noradrenaline storing vesicles in vas deferons from ox and rat was performed. Microsomal fractions were subjected to density gradient centrifugation. In rat, noradrenaline and dopamine β-hydroxylase were mainly present in the upper fractions of the gradient, which is consistent with the predominance of light (small dense-core) vesicles in this species. In ox, noradrenaline, dopamine and dopamine β-hydroxylase were found in the gradient in a bimodal distribution. This is consistent with the presence of about equal numbers of small and large dense-core vesicles in this species. On the other hand, chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity were only present in the dense (large dense-core) vesicle population. In order to study the capability of light and dense vesicles to synthesize noradrenaline we “pulse-labelled” ox vasa deferentia with [³H]tyrosine. Already 3.5 min after the pulse both types of vesicles contained [³H]noradrenaline and [³H]dopamine. During longer “chase” periods the amount of [³H]dopamine gradually declined.We conclude that dense (large dense-core) vesicles contain chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity whereas light (small dense-core) vesicles are devoid of these components. Both types of vesicles contain dopamine β-hydroxylase and can synthesize noradrenaline from dopamine under in vivo conditions.     

Electrophysiological evidence for an inhibitory accumbens-entopeduncular pathway in the rat

The effects of electrical stimulation of the nucleus accumbens (NA) on the activity of neurons of the entopeduncular nucleus (EP) were studied in the rat by extracellular single unit recordings. Many of the EP neurons were identified by antidromic activation from the lateral habenula (HBL). Stimulation of the NA induced inhibitions in 22.7% of the recorded EP cells. The data provide evidence for an inhibitory small projection from the NA to EP.