Lipids of nuclear fractions from neurons and glia of rat neocortex under conditions of artificial hypobiosis

Lipid contents were studied in tissue and nuclei isolated from neurons and glia of neocortex of rats under conditions of normothermia and in the state of artificial hypobiosis caused by hypothermia-hypoxia-hypercapnia. Compared to the neocortex tissue, both nuclear fractions were fivefold impoverished in phospholipids and cholesterol and strongly enriched with mono- and diglycerides and fatty acids. The nuclear fractions from neurons and glia contained similar amounts of phospholipids, and only the cardiolipin content in the neuronal nuclei was lower than in the glial nuclei. The state of artificial hypobiosis in rats led to an increase in the cholesterol/phospholipids ratio (mol/mol) in the nuclei from the neurons and glia; amounts of cholesterol and sphingomyelin in the nuclei from the glia were increased. The increases in the cholesterol and sphingomyelin contents and in the cholesterol/phospholipids ratio suggest an involvement of lipid-dependent signaling systems of the nuclei in the functional response of mammalian neocortex cells to artificial hypobiosis.     

Zinc can influence ornithine decarboxylase activity in rat thymus cells

Summary The thymus of young rats contained a high basal activity of ornithine decarboxylase (ODC). Treatment with zinc sulphate caused a slight increase of thymic ODC activity within 6 hours and a more marked enhancement (three-fold) in the spleen 24 h after treatment. In spite of the high activity of thymic ODCin vivo, ODC was not detectable in primary cultures of rat thymocytes, but was early and largely induced after treatment with Concanavalin A (Con A). The presence of 0.1 mM zinc in the medium increased the response of ODC to Con A. This effect of zinc in mitogen activated thymocytes may be due to the stabilization of ODC, which was found to decay with a half life of 65 min after the block of protein synthesis with cycloheximide. On the contrary in absence of zinc the half life of the enzyme was 40 min, as in the rat thymus in vivo.Zinc alone, at 0.1 mM concentration, did not affect ODC activity in resting thymocytes during the early times, but the metal was able to cause an increase of the enzyme activity after 4–6 days of culture. Other heavy metals such as mercury, cadmium and copper provoked a late increase of ODC activity, but their action was evident only at dosages which were toxic for the cells.     

Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation

We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a λgt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif ``IXCXE'. The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2–q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication. Received: 24 June 1998 / Accepted: 12 August 1998     

Mechanism of metabolic adaptation

The paper presents results of investigations on some mechanisms of metabolic adaptation in mammals. There are four sections in the tissue metabolic system of acid-base homeostasis: polyamines as factors of metabolic adaptation; significance of carbon dioxide for metabolic response formation in hypobiosis; polyamines metabolism in hypobiosis. Peculiarities of intermediate metabolism have been analyzed in animal tissues under the changes in H+, CO2 and HCO3- concentrations. Basing on a new interpretation of the experimental data and detected regularity in the metabolism, conclusion on the existence of a new acid-base homeostasis system in the tissues has been made. The results of polyamines metabolism investigations in the mammals under the stress have been described. The experimental data make us to believe that changes in polyamines synthesis and ODC activity in particular, is a part of stereotype nonspecific response to any stress impacts and one of the factors of cell metabolic adaptation. Some new data on mechanisms of formation and control of metabolic status of animals in the natural and artificial hibernation have been presented. The key idea is that in the state of hypobiosis the carbon dioxide (HCO3-) appears as a regulatory factor of metabolic adaptation, which is able to realize its action directly via affecting numerous biochemical events. The participation of polyamines in adaptive metabolic response to hybernation factors is suggested. Some peculiarities of ornithine decarboxilase and transglutaminase activity during the different stages of genuine and artificial hypobiosis have been demonstrated.     

Ornithine decarboxylase induction and polyamine levels in the kidney of estradiol-treated castrated male rats

Ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK) activities and polyamine concentrations on the kidneys of male castrated rats were studied following sc injection of estradiol. Estradiol caused an 11-fold increase in ODC activity 24 hours after administration. SAMDC activity doubled but TK activity decreased by two-thirds 2 days after estradiol treatment. The concentrations of polyamines, especially putrescine, showed sharp elevations 2 days following estradiol treatment, 1 day after the peak of ODC activity. The increase in ODC activity was suppressed by cycloheximide and by actinomycin D. Estradiol and diethylstilbestrol (DES), but not progesterone increased ODC activity. Estradiol suppressed ODC activities of liver, thymus, adrenal glands, testes and prostate. A specific estradiol-binding protein was demonstrated in the rat kidney. The dissociation constant (Kd) was 1.64 × 10^?10 M and numbers of binding sites were 31 fmoles/mg protein. Correlation between the binding of estradiol to the cytosol protein and elevation of ODC by estradiol was observed.     

Inverse relationship between adenosine deaminase and purine nucleoside phosphorylase in rat lymphocyte populations

The levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in rat lymphoid cell populations. The specific activities of the two enzymes in thymus, lymph node, spleen, and bone marrow were found in inverse proportion to each other. Thy mocytes had the highest ADA activity and the lowest PNP activity, whereas spleen and bone marrow lymphocytes had the lowest ADA activity (six- to sevenfold lower than thymocytes) and the highest PNP activity (fourfold higher than thymocytes). This reciprocal relationship was also found in cells of the T lymphocyte lineage at various stages of differentiation. These results suggest that specific stages of T-cell development may be characterized by the levels of the two enzymes, and that deficiencies of each of these enzymes might affect T cells at separate stages of differentiation.     

The characterization of the monoclonal antibody Th-10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG_2a and had κ light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G₁, G₂ and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characteize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or***     

Lipids in mammalian hibernation and artificial hypobiosis

Membrane lipids—phospholipids, fatty acids, and cholesterol—participate in thermal adaptation of ectotherms (bacteria, amphibians, reptiles, fishes) mainly via changes in membrane viscosity caused by the degree of fatty acids unsaturation, cholesterol/phospholipids ratio, and phospholipid composition. Studies of thermal adaptation of endotherms (mammals and birds) revealed the regulatory role of lipids in hibernation. Cholesterol and fatty acids participate in regulation of the parameters of torpor, gene expression, and activity of enzymes of lipid metabolism. Some changes in lipid metabolism during artificial and natural hypobiosis, namely, increased concentration of cholesterol and fatty acids in blood and decreased cholesterol concentration in neocortex, are analogous to those observed under stress conditions and coincide with mammalian nonspecific reactions to environmental agents. It is shown that the effects of artificial and natural hypobiosis on lipid composition of mammalian cell membranes are different. Changes in lipid composition cause changes in membrane morphology during mammalian hibernation. The effect of hypobiosis on lipid composition of membranes and cell organelles is specific and seems to be defined by the role of lipids in signaling systems. Comparative study of lipid metabolism in membranes and organelles during natural and artificial hypobiosis is promising for elucidation of adaptation of mammals to low ambient temperatures.     

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture

Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G₁ phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G₁ to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs.     

Tissue distribution of casein kinases

The activities of casein kinases 1 and 2 in cytosol fractions prepared from 12 different rat tissues were compared. Casein kinase activities were detected in all tissues examined. Total casein kinase activities of lung, spleen, testis, and thymus were much higher than those of skeletal muscle, cardiac muscle, and adrenal gland. When activities of casein kinases 1 and 2 partially purified from lung, spleen, testis, and thymus prepared from 5 rats were compared, both total and specific activities of these kinases in testis were higher than those in the other tissues. These results indicate that testis is the most suitable tissue in rats for large-scale purification of casein kinase 1 as well as casein kinase 2.     

Tissue distribution of casein kinases

The activities of casein kinases 1 and 2 in cytosol fractions prepared from 12 different rat tissues were compared. Casein kinase activities were detected in all tissues examined. Total casein kinase activities of lung, spleen, testis, and thymus were much higher than those of skeletal muscle, cardiac muscle, and adrenal gland. When activities of casein kinases 1 and 2 partially purified from lung, spleen, testis, and thymus prepared from 5 rats were compared, both total and specific activities of these kinases in testis were higher than those in the other tissues. These results indicate that testis is the most suitable tissue in rats for large-scale purification of casein kinase 1 as well as casein kinase 2.     

Studies on the relationship between cell proliferation and cell death: opposite patterns of SGP-2 and ornithine decarboxylase mRNA accumulation in PHA-stimulated human lymphocytes.

To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and sulfated glycoprotein 2 (SGP-2) were measured in human peripheral blood lymphocytes (HPBL) 2-6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, SGP-2, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of SGP-2 mRNA in quiescent HPBL, an early and progressive decrease of SGP-2 mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of SGP-2 and induction of ODC genes contribute for the cell entering the cell cycle.     

Effect of T-2 toxin on organ ultrastructure and organelle-specific enzyme activity in rats

The time-course of the ultrastructural changes and activities of 6 marker enzymes of subcellular particles (succinate dehydrogenase, beta-glucosidase, beta-N-acetylglucosaminidase, acid RNAse, glucose-6-phosphatase and 5'-nucleotidase) has been studied in the liver, spleen and thymus in rats administered T-2 toxin (mycotoxin produced by some Fusarium species). A pronounced difference in the effect of T-2 toxin on the organs has been found. In the liver, the toxin induced a destruction of rough endoplasmic reticulum membranes, reduced ribosome number and progressively decreased activities of most enzymes. In the spleen, early and significant ultrastructural disturbances of all the cell membrane components and simultaneous lysosomal activation were observed. The changes in the thymus were characterized by a fast development of cell hydratation, organelle swelling and necrosis of some thymocytes with parallel increase in repair processes, infiltration by phagocytes and a selective activation of lysosomal hydrolases in the end of experimental time (72 h.). The results obtained emphasize an importance of cellular and subcellular membrane alterations in the mechanism of T-2 toxin action.     

Multiple species of ornithine decarboxylase in rat tissues: Effects of dexamethasone

Several species of ornithine decarboxylase were separated by chromatography of rat thymus and kidney extracts on DEAE-Sepharose CL-6B. One major and one minor species were absent from thymus of rats two hours after hormone treatment but otherwise, the elution profile was identical to thymus from control animals. The elution patterns of ODC activity in kidneys of rats treated 2.5 or 5 hours before sacrifice with dexamethasone differ from that of control kidney and from each other. Enzyme from kidneys early after hormone treatment is eluted earlier than enzyme from control tissue, while at 5 hours, the enzyme is eluted much later than in the control. This suggests that the hormone-induced activity is subsequently modified.     

Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

Purine nucleoside metabolizing enzyme activities in mouse thymocytes at different stages of differentiation and maturation

The activities of the enzymes involved in purine nucleoside metabolism, adenosine deaminase (ADA), adenosine kinase (AK), purine nucleoside phosphorylase (PNP) and deoxycytidine kinase (deoxyCRK), were determined in mouse thymocytes at various stages of differentiation and maturation, and compared with those in other tissues. The thymocytes were characterized by high ADA and deoxyCRK activities with high ADA/AK and ADA/PNP ratios and low PNP/deoxyCRK ratio. In fetal thymocytes of 16 gestational days, ADA activity was lower, and PNP, AK and deoxyCRK activities were higher than those in the adult thymocytes. During differentiation of fetal thymocytes, ADA activity increased while PNP and AK activities decreased. DeoxyCRK activity decreased after birth. In spleen T lymphocytes, ADA and deoxyCRK activities were lower and PNP activity was about 2.5-fold higher than in the thymocytes. Thus the differentiation stages of T lymphocytes may be characterized by the absolute levels and the ratios of these enzymes.     

Natural cytotoxic autoantibody against thymocytes in spontaneously hypertensive rats

A strain of SHR rats, which spontaneously develops hypertension and periarteritis nodosa, had a decreasing number of rosette-forming T cells in their thymuses and a progressive decline in cellular immune functions by aging. They were found to produce natural thymocytotoxic autoantibody (NTA) detected by a complement-dependent cytotoxicity test. This autoantibody occurred from 1 month of age and throughout life; the incidence was more than 60% of SHR rats at any age. Thymocytes from all six rat strains tested showed similarly high sensitivity to NTA but none of the strains tested produced NTA except the SHR strain. Rosette-forming thymocytes of WKA rats, which were separated by Ficoll gradient, showed much higher cytotoxic sensitivity to NTA than did whole thymocytes and nonrosetting thymocytes. The cytotoxicity of NTA was weak or negative for spleen cells, lymph node cells, bone marrow cells, and blood lymphocytes of WKA rats. However, the cytotoxic activity of NTA was completely absorbed with the thymus, spleen, lymph node cells, and brain homogenates and was partially absorbed with bone marrow cells, but not with liver and kidney homogenates. NTA in SHR rats was an IgM-globulin as determined by sensitivity to 2-ME treatment and by Sephadex G-200 column chromatography. These results suggest that NTA is responsible for the selective suppression of T-cell functions in SHR rats.     

Prolactin receptor gene expression in rat splenocytes and thymocytes during oestrous cycle, pregnancy and lactation

Much evidence suggests that prolactin (PRL) has an immunoregulatory function. Part of this evidence is that the receptors for PRL are present on lymphocytes. Probably the effects of PRL on cells of the immune system depend on the level and specific forms of PRL-R present on the target cells. Therefore, PRL-R expression at both protein and mRNA levels was examined during oestrous cycle, pregnancy and lactation using Western blotting and PCR analysis. Antibody to the long form of PRL-R detected 84 and 42 kDa protein bands in the spleen but only 84 kDa band in the thymus. The expression pattern of these two protein bands was different in the spleen, suggesting that these two isoforms of PRL-R long form are differentially regulated by the hormones of oestrous cycle. In addition, depending on the tissue, the level of mRNA for the short and long forms of PRL-R showed a significant change at different stages of oestrous cycle. Moreover, 42 and 84 kDa PRL-R bands were detected in both spleen and thymus throughout the pregnancy and lactation; however, the expression pattern of 84 kDa protein band was different between tissues. This finding suggests that each tissue exhibits differential response to hormones which affect PRL-R content.     

脾虚证小鼠胸腺细胞周期和免疫细胞化学的实验研究

本文用ＦＣＭ和免疫细胞化学技术检测了小鼠胸腺细胞的活动周期,Ｂｒｄｕ＋细胞和表达Ｔｈｙｌ．２抗原的Ｔ细胞。小鼠包括对照组,利血平诱发的脾虚组,自然恢复组和健脾汤治疗后复健组。ＦＣＭ和免疫细胞化学结果显示,脾虚组小鼠Ｇ１期细胞堆积,Ｓ期和Ｇ２＋Ｍ期细胞减少。同时胸腺内Ｂｒｄｕ＋细胞和Ｔ细胞也相应减少。经过健脾汤治疗后,除Ｇ１期细胞外,Ｓ期和Ｇ２＋Ｍ期细胞,Ｂｒｄｕ＋细胞和Ｔ细胞均显著增加,并且几乎达正常水平。但是自然恢复组在恢复细胞活动周期正常化,以及恢复Ｂｒｄｕ＋细胞和Ｔ细胞方面比复健组缓慢。说明健脾汤对脾虚小鼠的胸腺细胞有促进ＤＮＡ合成和细胞增殖功能。