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.     

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.     

Age-dependent alterations in lipids and function of rat heart sarcolemma

The objective of the present study was to examine the effect of age on heart sarcolemma structure and function. Sarcolemmal fractions were prepared from hearts of young (1–1.5 months) and adult (10–12 months) rats and assayed for marker enzyme activities. The membrane fractions were found to be devoid of other cellular organelles upon examination by electron microscopy. They were enriched with 5′-nucleotidase and devoid of succinate dehydrogenase activity. The only age-related lipid compositional changes noted in these membranes were changes in the fatty acid composition of membrane phospholipids with increasing age. Most changes were detected in phosphatidylcholine and phosphatidylethanolamine with very little alteration of sphingomyelin and phosphatidylserine plus phosphatidylinositol fatty acids. Polyunsaturated fatty acids, especially 18:2 and 20:4, were decreased with saturated fatty acids increased in membrane phosphatidylcholine and phosphatidylethanolamine fractions as the animal develops. There was a decrease in the specific activities of(Na⁺ + K⁺)-ATPase and 5′-nucleotidase of these membranes with age. On the other hand, membrane (K⁺)-p-nitrophenylphosphatase was not affected by age.     

Interferon action against reovirus: activation of interferon-induced protein kinase in mouse L929 cells upon reovirus infection

Experiments are presented which indicate that reovirus infection results in an activation of the interferon (IFN)-induced protein kinase in mouse L929 cells. This is indicated by (i) the phosphorylation in vivo of a 67,000 M_r (67K) polypeptide, which is characteristic of the IFN-induced protein kinase, within a few hours after reovirus infection of IFN-treated mouse L929 cells, and (ii) the phosphorylation in vitro of the 67K polypeptide as well as the α-subunit of exogenously added initiation factor eIF-2 in extracts of IFN-treated reovirus-infected cells without the addition of double-stranded RNA which is required in similar extracts from uninfected cells. In NIH 3T3 cells, which are deficient in (2′,5′)oligoadenylate-activated endonuclease, the replication of reovirus is inhibited by IFN treatment, though EMC virus replication is insensitive. It is suggested that this kinase activation observed upon reovirus infection may play a role in the antiviral action of IFN against reovirus.     

Kinetics of parvovirus replication,cytopathic effects,and mitotic arrest in synchronized bovine fetal spleen cells

Initiation of autonomous parvovirus replication depends on the S phase of host cells actively traversing the cell cycle. The parvoviruses Lu III and H-1 inhibit synchronized cells from entering mitosis, implying that parvoviruses rapidly shut down cell cycle traverse during G2 phase. Bovine parvovirus (BPV) did not inhibit the entry into mitosis of hydroxyurea synchronized bovine fetal spleen cells. Mitotic indexes of infected cultures were as much as 60-fold higher than those of mock-infected controls. Mitosis in control and infected cultures peaked at 10 hr after infection corresponding to the end of the BPV eclipse period. Cytopathic changes, including morphological alteration of mitotic chromosomes, were detected in mitotic cells from infected cultures by light and electron microscopy. Arrest of BPV-infected cells in mitosis may explain these results. Not all infected cells were killed in mitosis, since some developed intranuclear inclusions and became pycnotic as nucleated, interphase cells. Inclusion formation was coincident with viral morphogenesis in interphase nuclei at 16 to 18 hr postinfection, late in the viral replication cycle. The cell cycle stage at which parvovirus-infected cells are arrested and cytopathic events ensue may be determined by the cellular progression rate from S phase through G2 and M.     

Identification of a second protein (M2) encoded by RNA segment 7 of influenza virus

Recent analyses of the nucleotide sequences of RNA segment 7 of influenza virus from two strains of infuenza A virus (PR/8/34 and Udorn/72) have shown that in addition to the reading frame coding for the membrane protein (M₁) there is a second overlapping open reading frame that could code for 97 amino acids [Winter and Fields (1980). Nucleic Acids Res.8, 1965–1974;Allen et al. (1980). Virology107, 548–551;Lamb and Lai (1981). Virology112, 746–751]. We have identified such a protein (M₂) in infected cells and shown that it is synthesized from a separate mRNA. Hybrid-arrested translation experiments and the use of recombinant viruses of defined genome composition have demonstrated by both biochemical and genetic approaches that protein M₂ is coded for by RNA segment 7. Peptide mapping studies have shown that M₁ and M₂ are distinct proteins, and the peptide maps can be correlated with the nucleotide sequences coding for M₁ and M₂.     

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     

Histochemical assessment of nuclear protein content and enzyme activity in confluent nonarrested and low-serum arrested WI-38 fibroblasts at mid and late population doubling levels

Cultures of WI-38 fibroblasts maintained in low-serum medium have little mitotic activity. Such arrested cultures can serve as a useful adjunct to the normal-serum proliferating fibroblast model system of senescence since the reduced cycling activity more accurately reflects in vivo conditions. Normal-serum plateau phase cultures and 17-day low-serum arrested plateau phase cultures were examined at both mid and late population doubling levels (PDL). Acidic, basic and total nuclear protein contents were determined by staining with toluidine blue O, alkaline fast green and acid fast green, respectively. Succinate dehydrogenase and glucose-6-phosphate dehydrogenase were localized with the nitroblue tetrazolium staining reaction. Quantification of individual cell staining was done on a Vickers M85 microdensitometer. Low-serum arrested cells at both mid and late PDL showed a significant increase in acidic protein staining and a decrease in basic protein staining when compared with normal-serum nonarrested cells. Enzyme activities in arrested cells were lower than nonarrested cells at mid PDL but equivalent to nonarrested cells at late PDL. This study demonstrates that the use of selected quantitative histochemical staining in combination with cytophotometry can be employed to differentiate arrested from nonarrested fibroblast populations as well as mid PDL from late PDL cultures.     

Induction of deoxythymidine kinase activity in several mammalian cell lines after infection with six different strains of equine herpesvirus type 1 (EHV-1)

Infection of cells derived from three different mammalian species with any one of six strains of EHV-1 resulted in a 5- to 10-fold increase in the deoxythymidine kinase (dTK) activity when assayed in the presence of 100 μM dTTP. Increased dTK activity was also demonstrated in liver homogenates prepared from EHV-1-infected Syrian hamsters. Antibody elicited against the EHV-1 dTK induced in horse cells neutralized the enzyme activity induced by EHV-1 infection of horse, mouse, hamster, and monkey cells but not the dTKs of uninfected, nonequine cells. EHV-1 induction of dTK was confirmed by demonstration of the inability of the virus strains to replicate in the presence of arabinosylthymine (ara-T) in cells which themselves were totally resistant to the drug. Mutants of EHV-1 resistant to 5-bromodeoxyuridine and ara-T and lacking the ability to induce dTK activity (dTK⁻) were also isolated. Electrophoretic analysis of cell extracts in polyacrylamide gels revealed a new peak of dTK activity (R_f = 0.25) after EHV-1 infection of some cell types (e.g., dTK⁻ 3T3 cells, owl monkey kidney cells, etc.). Other cell types, whose cytosol dTK migrated with an R_f (0.25) identical with that of the EHV-1 enzyme, contained no new electrophoretic peak of dTK activity after infection. In these latter infected cells, however, the dTK activity with an R_f value of 0.25 was partially inhibited by anti-(EHV-1) serum, partially resistant to 100 μM dTTP, and displayed activity even with CTP as a phosphate donor, suggesting that, in these particular cell types, the EHV-1-induced dTK and the host cell cytosol dTK co-electrophorese. These results indicate that infection of cells with all strains of EHV-1 results in the induction of a new, virus-coded dTK activity.     

Genetically determined conidial longevity is positively correlated with superoxide dismutase, catalase, glutathione peroxidase, cytochrome c peroxidase, and ascorbate free radical reductase activities in Neurospora crassa

Aging of post-mitotic cells, the conidia, of Neurospora crassa is defined as the time-dependent loss of viability under a constant laboratory environment which probably resembles the organism's tropical habitat; namely, at 30 degrees C, 85-100% relative humidity under white light. Median lifespan is defined as the age at which survival of a conidial population has declined to 50% of that of a fully viable population at birth. A collection of short (age-) and long-lived (age+) mutants were previously selected from the wild-type whose median lifespan is 22 days. Thus, five groups of strains with distinct lifespans of 7, 22, 36, 50 and 60 days were defined. The purposes of the present investigation were to determine if the activities of anti-oxygenic enzymes are correlated with lifespan and to elucidate the function of the cellular longevity determinant genes. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) were highly-correlated with lifespan; whereas glutathione reductase and non-specific peroxidase activities were not correlated. The short-lived mutants were also deficient in cytochrome c peroxidase (CPX) and ascorbate free radical reductase (AFR), but not deficient in dehydroascorbate reductase. (These latter three enzymes were not examined in age+ mutants.) By isoelectric focusing analysis, the deficiencies of SOD, CAT, and GPX activities of age- mutants were defined in terms of specific isozymes. The mutants were specifically deficient in a cyanide-resistant mitochondrial isozyme of SOD. Sixteen age- genes, called the age-1 complex, were previously mapped on one arm of the seven chromosomes. On the basis of mapping and complementation data, it was inferred that the genes are spatially and functionally redundant. The hypothesis of functional redundancy is also supported by the enzyme data. Of seven mutants examined, representing seven of the age- genes, all were deficient in SOD, CAT and CPX, and six were deficient in AFR. Of four mutants examined, representing four of the genes, all were deficient in GPX. The results indicate a molecular basis for the previously observed photosensitivity of the mutants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental murine leprosy: a biochemical study emphasizing lysosomal enzyme changes in vivo and enzyme secretion by macrophages in vitro

This study was undertaken to identify biochemical alterations in serum, lymphoid organs, and peritoneal macrophages (PM) which reflect the histopathology of experimental Mycobacterium lepraemurium (MLM) infection in mice. A significant increase of acid phosphatase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and lysozyme was found in serum, spleen, and liver homogenates of mice infected intraperitoneally (ip) with MLM. PM from infected mice showed a substantially greater rate of secretion of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and acid phosphatase than PM from normal mice. There was, however, no significant difference in the ability of PM from BALB/c and C57BL/6 mice to secrete such enzymes in vitro. There was also a significant increase in all these enzymes in PM in the early stage of infection but they dropped to values lower than normal in the advanced stage of infection despite the fact that such cells increased in size and protein content as the infection progressed. Infected mice were also found to have progressively elevated levels of serum lactic dehydrogenase, glutamic oxaloacetic, and glutamic pyruvic transaminases which indicated damages of hepatocytes and other tissues. Values of other blood components were also reported. Both BALB/c and C57BL/6 strain of mice, which are susceptible to the ip route of MLM infection, showed an indistinguishable pattern of biochemical alterations as reflected by their similar histopathological changes in various organs. BALB/c mice, which are still susceptible to subcutaneous (sc) route of infection showed similar characteristic changes in various serum components as before. In contrast, C57BL/6 mice, which are resistant to MLM infection sc, showed insignificant alterations in most of these biochemical parameters.     

Organ pattern of age-related changes in the aminoacyl-tRNA synthetase activities of the mouse

The specific activities of 17 aminoacyl-tRNA synthetases from liver, lung, heart, spleen, kidney, small intestine and skeletal muscle of young (2 months) and aged (39 months) female Han:NMRI mice were determined under standard conditions of sample preparation and assay. The average reduction of total activity during ageing is 70% for liver, 50% for lung and spleen, nearly 40% for heart and kidney and nearly 20% for intestine and skeletal muscle. Detailed comparison reveals no general, but an organ-specific pattern.

Aminoacyl-tRNA synthetases were, furthermore, found to be ribosome-associated in higher proportions in liver tissue from aged mice.     

Lysosomal and nonlysosomal proteolytic activities in experimental diabetic cardiomyopathy

The role of cardiac lysosomal and nonlysosomal protease alterations in the development of the cardiomyopathy that occurs in genetically diabetic C57BL/KsJ db/db mice has been examined. The db/db mice and age-matched controls were sacrificed between 7 and 24 weeks of age. Cathepsin D activity, myofibrillar alkaline protease (MAP) activity (including serine protease activity), and Ca2+-activated protease activity were determined by using [3H]acetyl-casein as substrate. There is a significant decrease in cathepsin D, MAP, and serine protease activities in the myocardium of 7- to 20-week old diabetic mice with a rebound of these activities toward normal levels by 24 weeks of age. Cathepsin D and MAP activities are inversely related to heart weight in diabetic mice with the higher levels being recorded in association with the most pronounced decrease in heart weight. In contrast, Ca2+-activated protease activity in the hearts of diabetic mice does not differ significantly from controls throughout the period of observation. The results suggest that both lysosomal cathepsin D and nonlysosomal MAP may mediate the accelerated cardiac muscle degradation that occurs in the late stage of diabetic cardiomyopathy in the db/db mice.     

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.     

Evidence for different receptor sites in mouse spleen cells for the Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins

Liposomes were reconstituted from phosphatidylcholine and Sendai virus glycoproteins HN or F and their interaction with mouse spleen cells was studied. Both the HN and F liposomes were able to stimulate chemiluminescence (CL), indicating that the glycoproteins were able to interact with the cell membrane independently of each other. The induction of CL in cells which had been pretreated with liposomes by monoclonal antibodies to either HN or F demonstrated that HN and F bind to the cells independently. The presence of F liposomes on the cell surface was also confirmed by immunoelectron microscopy. Cells pretreated with HN and F liposomes revealed a different pattern of CL when challenged with intact virus or the calcium ionophore A23187 indicating that HN and F bind to different receptor sites.     

Autoregulation of histamine release in brain by presynaptic H3-receptors

Regulation of histamine release was studied mainly on brain slices prelabeled with L-[3H]-histidine and depolarized by increased extracellular K+ concentration or veratridine in a non-superfused system. The released 3H-labeled amines, isolated by ion-exchange chromatography from a large excess of 3H-labeled precursor consisted by more than 95% of unchanged [3H]histamine. Exogenous histamine reduced the release of neosynthesized [3H]histamine via stimulation of previously characterized H3-receptors whereas it did not modify the 3H-labeled amine release from slices prelabeled with preformed [3H]histamine. The maximal inhibitory effect of exogenous histamine progressively diminished as the strength of the depolarizing stimulus or the external Ca2+ concentration were elevated. On the contrary H3-receptor antagonists like impromidine or burimamide enhanced the depolarization-induced release of [3H]histamine, an effect which was particularly marked when slices were loaded with histamine by preincubation with [3H]histidine in high concentration. These results suggest that the inhibition of [3H]histamine release by exogenous histamine acting via H3-receptor stimulation is mediated by a restricted access of Ca2+ and that its extent is influenced by the degree of autostimulation by endogenous histamine as well as, possibly, by actual internal Ca2+ concentration. In addition the decrease in external Ca2+ concentration shifted rightwards the concentration-response curve to histamine. The autoinhibitory effect of exogenous histamine was found on slices from various regions, known from lesion studies to contain terminals of extrinsic histaminergic neurons. It did not apparently involve interneurones, not being prevented in slices in which the traffic of action potentials was blocked by tetrodotoxin. It also remained unaffected in striatal slices in which the neuronal cell-bodies were selectively destroyed by prior local infusion of kainic acid. Finally exogenous histamine inhibited [3H]histamine release from depolarized synaptosomes of rat cerebral cortex, with an EC50 value similar to that found with slices and was antagonised by impromidine with an apparent Ki value similar to that displayed at H3-receptors. It is concluded that histamine modulates its own release from cerebral neurones by interacting with H3-presynaptic autoreceptors and via mechanisms similar to those previously evidenced on other aminergic systems.     

Isolation of an avian sarcoma virus-specific protein kinase from virus particles

Avian sarcoma viruses (ASV) of the Schmidt-Ruppin strain (SR) contain a protein kinase activity which specifically phosphorylates the IgG of sera from tumor-bearing rabbits (TBR). The amount is comparable with that from transformed cells. The activity is thermolabile in two mutants with a temperature-sensitive lesion in the sarcoma (src) gene. Ion-exchange column chromatography on DEAE-cellulose and phosphocellulose allowed a 250-fold purification of enzymatically active protein kinase with a molecular weight of 60,000 (60K). It phosphorylated casein and the heavy chain of IgG of TBR sera but not of control sera. Phosphorylation of casein could be completely inhibited by TBR serum and resulted in phosphorylation of IgG. Purification of the protein kinase from a mutant virus, OS122, and its wild-type SR-D revealed a threefold higher thermolability for the mutant enzyme. Partial proteolytic digest of the [³⁵S]methionine-labeled 60K protein obtained from the phosphocellulose column by immune precipitation was indistinguishable from that of pp60^STC precipitated from SR-D-transformed cells.     

An immunological hypothesis for plasma protein catabolism

A selective mechanism of plasma protein catabolism is suggested, based on three main assumptions: (1) plasma proteins are submitted to molecular ageing thus achieving “modified” forms after a given time period; (2) the system recognizing the “modified” molecules consists of preformed physiological autoantibodies; (3) the elimination of the immune complexes formed between “modified” proteins and the corresponding autoantibodies takes place via binding to Fc receptor bearing cells.