Crystalline desoxyribonuclease; digestion of thymus nucleic acid; the kinetics of the reaction

A study was made of the enzymatic properties of crystalline desoxyribonuclease. The general effect of the crystalline enzyme on its specific substrate, thymus nucleic acid, was found to be essentially the same as described by previous workers for the digestive action of crude preparations of the enzyme. The digestive action consists mainly in splitting thymus nucleic acid into fragments approaching the size of tetranucleotides. The digested nucleic acid is diffusible through collodion or cellophane membranes and is non-precipitable with strong acid, alcohol, or proteins. The digestion of thymus nucleic acid by desoxyribonuclease is accompanied by the liberation of one atom equivalent of free acid per four atoms of nucleic acid phosphorus. Crystalline desoxyribonuclease acts very slowly, if at all, in the absence of magnesium (or manganese) ions. The optimal concentration of magnesium ion required increases with the increase in concentration of the substrate but is independent of the enzyme concentration. The optimal pH range for the action of crystalline desoxyribonuclease is 6.0 to 7.0. A study was made of the kinetics of the digestion of thymus nucleic acid as manifested mainly by the gradual formation of acid-soluble split products. At low concentrations of nucleic acid, the process approximates closely a reaction of the first order, the unimolecular constant being independent of the concentration of desoxyribonuclease in the digestion mixture. At relatively higher concentrations of substrate, however, the initial rate of reaction decreases rapidly with the increase in concentration of substrate, and the reaction as a whole is represented by non-symmetric S-shaped curves apparently too complicated for a simple rational interpretation.     

The effect of thymus extracts on phosphorus compounds in muscle and serum, and on serum calcium

1. Thymectomy in young rabbits decreased the ATP content and increased the inorganic phosphate content of skeletal muscle. The serum calcium content was decreased, whereas the inorganic phosphate content was increased. 2. The administration of a lipid fraction (TL) or protein fractions (CIF and TP) of thymus extracts to thymectomized rabbits in short-term experiments increased the ATP content of muscle and decreased the inorganic phosphate contents of muscle and serum. Serum calcium content was increased. 3. The action of the thymus extract TP was specific only on the phosphate compounds, since the increase in serum calcium concentration was also caused by the control extract from muscle. The action of the extract TL is not specific, being paralleled by the action of a control extract from muscle.     

L-tyrosine: 2-oxoglutarate aminotransferase induction by hydrocortisone in the thymus of the white RAT

Hydrocortisone hemisuccinate within 4 hours after in vivo administration produced an increase in precursor incoporation into rat thymus RNA and proteins in the whole animal. From these results, together with information obtained from measurements of the tyrosine aminotransferase activity and the action of mitomycin C administered one hour before the injection of hydrocortisone, it can be concluded that the increase in tissue level of the enzyme, consequent to hydrocortisone treatment, results from an increased rate of biosynthesis of the enzyme, which participates in the catabolic processes of proteins in glucocorticoid sensitive thymus cells.     

Effect of gamma-radiation and mitochondrial apoptogenic factors on nuclear protease activity

An increase in protease activity was shown in thymus nuclei of rats exposed to gamma-radiation. The activation of histone-specific proteases depended on the duration of postradiation period. Also, it was revealed that incubation of thymus nuclear with the intermembrane fraction of liver mitochondria caused degradation of histones and nonhistone nuclear proteins, as well as internucleosomal fragmentation of DNA. Simultaneously, nuclear proteases tightly bound to histones and specifically cleaving histones were observed to be activated by apoptogenic factors of the mitochondrial intermembrane fraction. Probably, the apoptogenic action of gamma-radiation involves not only a direct DNA damage that induces activation of DNA-dependent proteases but also an indirect component: destructive alterations in mitochondria leading to the exit of apoptogenic factors from the intermembrane space.     

In vitro modulation of mouse natural killer (NK) cell activity by the serum thymic factor (FTS)

Previous studies indicated that the serum thymic factor (FTS) could modulate in vivo the level of splenic natural killer (NK) cell activity in mice. The present report shows that such an effect is also observed after a short term in vitro incubation of the effector cells with FTS. The regulatory effects of FTS result in an increase or a decrease of the splenic NK cell cytotoxicity depending upon the age and the mouse strain. Furthermore, FTS is able to enhance the NK cell activity of thymus and bone marrow cells which are known to be weakly reactive in NK cytotoxicity. Depletion experiments demonstrated that the FTS-induced increase of NK cell activity was not mediated by Thy 1+ cells nor macrophages, thus suggesting a direct action of FTS on the effector cells. Comparative studies using other thymic hormones revealed similar patterns of reactivity. These results favor the hypothesis of a close relationship between the thymus and NK cells.     

8-Chloroadenine: a novel product formed from hypochlorous acid-induced damage to calf thymus DNA

Hypochlorous acid (HOCl) is formed by the action of the enzyme myeloperoxidase on hydrogen peroxide and chloride ions. It has been shown to be highly bactericidal and cytotoxic by a variety of mechanisms, one of which, may be the modification of DNA. Previously we have demonstrated by GC-MS analysis that exposure of calf thymus DNA to HOCl causes extensive pyrimidine modification, including 5-chlorocytosine formation. Using GC-MS analysis, we now demonstrate the formation of an additional chlorinated base product, 8-Cl adenine. The addition of 50 μM HOCl was sufficient to produce a significant increase in this product. The reaction of HOCl with adenine in calf thymus DNA was shown to be rapid with the reaction complete after 1 min. pH-dependence studies suggest HOCl rather than its conjugate base (OCl-) to be responsible for 8-Cl adenine formation. Other commercially available chlorinated base products, 6-Cl guanine or 2-Cl adenine were not detected. Therefore, 8-Cl adenine might prove a useful biomarker for studying the role of reactive chlorine species (RCS) during inflammatory processes.     

Mechanism of action of a mammalian DNA repair endonuclease

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.     

The immunoproliferative response of mice to intravenously or subcutaneously injected BCG

Summary Intravenous injection of BCG caused (1) a transient thymic epithelial hyperplasia with increase of PAS-positive cells in the cortex and medulla which showed the pronounced secretory activity of a substance which could be histochemically identified as an acid mucopolysaccharide; (2) an equally transient increase in the number of pyroninophilic lymphocytes with increased polyribosome content of the cells and mitoses in the thymic cortex; this reached a peak on day 6 following the injection but was unassociated with an increase in thymic weight; and (3) a systemic granulomatous histiocytic reaction in the liver, spleen, lungs, and lymph nodes, but not in the thymus, bone marrow, or Peyer's patches. The significance of the thymic epithelial changes is not clear but it did coincide with increased pyroninophilia and mitotic activity of the thymic cortical cells, suggesting a possible interaction between this secretory product and the thymic cortex. Comparing the thymic changes with the thymus of other animals of the same species injected with i.v. or i.p. LPS, i.v. or s.c. HIU II fraction of BCG, i.v. pertussis vaccine, i.p. complete or incomplete Freund's adjuvant, and killed at the same planned intervals after the injection of the adjuvants, BCG proved to have a unique action on the thymus with regard to both lymphocytic and epithelial changes. Hepatic, pulmonary, and splenic histiocytic granulomas were observed only in those animals injected intravenously with BCG.     

Specificity of the effect of growth hormone on DNA concentration in the nuclei of the lymphoid cells of hypophysectomized rats]

The effect of growth hormone on the DNA content in the nuclei of the thymus, spleen and the lymph node lymphocytes was studied by means of cytophotometry. In hypophysectomized rats the growth hormone increased the DNA content in the nuclei of the middle lymphocytes of these organs without altering its amount in the small lymphocytes. Thymus lymphocytes were the most sensitive to the hormone action. The DNA content in the nuclei of these cells increased as soon as one hour after the administration of the hormone; in 4 hours it reached the maximum. Other hormones with an anabolic effect (insulin, thyroxin, testosterone), induced no elevation of DNA in the thymocyte nuclei at that period of time. A conclusion was drawn on the high tropicity of the growth hormone to the cells of the lymphoid organs and particularly to the thymocytes (middle lymphocytes of the thymus).     

The annual involution and regeneration of the thymus in hibernating animals and perspectives of its studies in gerontology and stem cell proliferation

Data on a unique phenomenon of annual involution and neogenesis of thymus gland in hibernating animals are reviewed. In accordance with morphological findings, the annual thymus involution in hibernating animals is close to the age-dependent thymus involution occurring in all mammals once in a lifetime. In opposite, thymus involution in hibernating animals is totally different from the accidental involution. During hibernation, the thymus tissue is substituted by the brown fat tissue. In the spring, thymus gland neogenesis stats with intensive growth of epithelial tissue followed by lymphocyte infiltration and exhaustion of brown tissue. Morphological changes in the thymus gland within the annual cycle were compared with seasonal dynamics of structural and functional changes in peripheral lymphoid organs (spleen, lymphoglandular, peritoneal fluid). A general regularity was observed involving a decreased functional activity of immune cells in autumn, its sharp depression during winter hibernation, and obvious increase in summer with the onset of a season of animal activity. It is supposed that a sharp increase in the tumor necrosis factor (TNF) production observed during short-term awakenings in winter may serve an important link in this unique immune adaptation mechanism. The season changes in cellular TNF secretion suggest a mobilization of protective resources in hibernating animals in autumn and winter, i.e. in seasons when the thymus gland activity is depressed. The annual involution of thymus gland cannot be related to droppings in the environmental or body temperatures, as it comes long before their fall. Additionally, it is not related to ageing, as it occurs already in young hibernating animals. The role of hormones, including melatonine and corticosteroids, in mechanisms regulating thymus gland involution in hibernating animals is discussed.     

8-Chloroadenine: a novel product formed from hypochlorous acid-induced damage to calf thymus DNA

Hypochlorous acid (HOCl) is formed by the action of the enzyme myeloperoxidase on hydrogen peroxide and chloride ions. It has been shown to be highly bactericidal and cytotoxic by a variety of mechanisms, one of which, may be the modification of DNA. Previously we have demonstrated by GC-MS analysis that exposure of calf thymus DNA to HOCl causes extensive pyrimidine modification, including 5-chlorocytosine formation. Using GC-MS analysis, we now demonstrate the formation of an additional chlorinated base product, 8-Cl adenine. The addition of 50 μM HOCl was sufficient to produce a significant increase in this product. The reaction of HOCl with adenine in calf thymus DNA was shown to be rapid with the reaction complete after 1 min. pH-dependence studies suggest HOCl rather than its conjugate base (OCl-) to be responsible for 8-Cl adenine formation. Other commercially available chlorinated base products, 6-Cl guanine or 2-Cl adenine were not detected. Therefore, 8-Cl adenine might prove a useful biomarker for studying the role of reactive chlorine species (RCS) during inflammatory processes.     

胸腺对雌性大鼠肝脏抗氧化功能的影响及其与细胞免疫、性激素水平的关系

雌性成年大鼠切除胸腺后,其肝脏脂质过氧化产物丙二醛增多,还原型谷胱甘肽(GSH)含量减少,微粒体和线粒体膜流动性降低。隔日1次皮下注射自猪胸腺提取的胸腺因子D(TFD)2mg/kg共3月,可逆转去胸腺大鼠上述指标的变化。为了探讨胸腺影响肝脏抗氧化功能的中间途径,还测定了脾T淋巴细胞增殖反应及血浆性激素水平。结果表明,去胸腺大鼠脾脏T淋巴细胞增殖反应减弱、血浆雌二醇/睾酮比值下降;给予TFD可逆转其变化。实验结果提示,胸腺对大鼠肝脏抗氧化功能的影响可能与胸腺-内分泌-免疫网络有关。     

Thymic structural changes in relation to seasonal cycle and testosterone administration in wall lizard Hemidactylus flaviviridis (Ruppell)

Light microscopic and ultrastructural studies of thymus in wall lizard showed remarkable season dependent structural changes. In winter, the thymus was involuted and its cortico-medullary differentiation was not distinct. Thymocytes were sparsely distributed. The epithelial cells exhibited atrophic features such as an appreciable decrease in the nuclear-cytoplasmic ratio and accordingly reduction in cell organelles. The reconstruction of thymus commenced during spring and it became fully developed with marked delineation of cortico-medullary regions during summer. The thymus was then densely populated with thymocytes and epithelial cells showed voluminous cytoplasm having numerous cell organelles. The thymus regression started again by the beginning of autumn. The results suggest that the thymic development in wall lizard have inverse relationship with the androgen level, as the testicular steroidogenic activity was seen maximum during winter and least in summer. This assumption gets support by castration and testosterone replacement experiments. Castration of lizards during winter resulted in profound development of thymus with an appreciable increase in thymocytes mainly in the cortex region . The cortex became delineated from the medulla. Following testosterone treatment, the thymus underwent regression and was comparable to testis-intact lizard's thymus during winter season. After withdrawal of testosterone treatment, the thymus exhibited dense lymphoid and thymocyte population with a demarcation of cortico-medullary regions and sub-cortical region was regenerated.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N1-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N1-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N1-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N1-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

AP液抗衰老作用的实验研究

Ｄ－半乳糖皮下注射致Ｗｉｓｔａｒ大鼠亚急性衰老模型,观察ＡＰ液对大鼠衰老指标的影响。实验结果显示,ＡＰ液能明显增加胸腺重／体重的比值,提高血清ＳＯＤ活性和降低血清ＬＰＯ活性。ＡＰ液抗衰老组血清ＳＯＤ活性比衰老模型组增加２５．２９％,血清ＬＰＯ活性下降６．２４％,提示ＡＰ液可能具有抗衰老作用。     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coli O55 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coli O111 or that by Boivin's method from E. coli O55 was similar to that of CPS-K. It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.     

Gangliosides as markers of cortisone-sensitive and cortisone-resistant rabbit thymocytes: characterization of thymus-specific gangliosides and preferential changes of particular gangliosides in the thymus of cortisone-treated rabbits

Neutral glycosphingolipids and gangliosides in rabbit thymus, spleen, bone marrow, and erythrocyte ghosts were analyzed by conventional chemical and enzymatic procedures and negative ion fast atom bombardment mass spectrometry (FABMS). Thymus gangliosides showed a characteristic composition. Major gangliosides comprising 75% of the total thymus gangliosides were sialosyl lacto-N-neo-tetraosyl- and sialosyl lacto-N-nor-hexaosylceramides containing NeuGc and palmitic acid. These major thymus gangliosides were not detected in spleen, bone marrow, or erythrocytes, whereas GD1a, which was not present in the thymus even in a trace amount, was present in spleen and bone marrow. In addition, the major gangliosides in rabbit thymus were preferentially reduced when an animal was given an intraperitoneal injection of cortisone acetate, as found on analysis 48 h later. The decrease was accompanied by a concomitant increase in NeuAc-containing GM3 with longer chain fatty acids.     

Inhibition of DNA polymerase-alpha and -beta of calf thymus by 1-beta-D-arabinofuranosylcytosine-5'-triphosphate.

1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP), an active form of a inhibitor of DNA replication, 1-beta-D-arabinofuranosylcytosine (araC) was tested for its inhibitory action on the DNA polymerase-alpha and -beta (EC 2.7.7.7) purified from calf thymus. The reaction of DNA polymerase-alpha was shown to be more sensitive to the inhibition by araCTP than that of DNA polymerase-beta. The mode of the inhibition by araCTP was competitive to dCTP in the reaction catalysed by either DNA polymerase-alpha or -beta. The Ki value of DNA polymerase-beta for araCTP was 32 micron; eight times higher than that of DNA polymerase-alpha (4 micron) for this inhibition.