The Activity of 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase in Rat Tissues

Abstract: The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was measured in 14 rat tissues and in subcellular fractions of rat liver by a sensitive fluorometric method, using cyclic NADP as substrate. CNP activity in brain (339 μmol/h/mg protein) was fourfold that of the sciatic nerve. The activities in tissues outside the nervous system ranged from a low of 0.42 μmol/h/mg protein in the unwashed red blood cell to a high of 9.96 in the spleen. The activity was highest in tissues containing cells with membranes capable of undergoing transformation and elaboration (spleen and thymus) and low in those in which the cell membranes are morphologically stable (muscle and red cell). The enzyme was found in all major liver subtractions, with the highest activities in the microsomal and nuclear fractions. Despite the large difference in the maximal velocities of CNP in brain and liver, the affinity of the liver enzyme for the substrate ( k _m) was similar to that of brain enzyme. Brain CNP was stable over a 48-h postmortem period.     

Immunological effects of recombinant feline interferon-omega (KT-80) administration in the dog

The immunological effects of recombinant feline interferon-omega (rFeIFN-omega ; KT-80, Toray) were examined on administration to healthy dogs. The activities of whole blood cells, macrophages, and natural killer cells were enhanced. Moreover, the whole blood activity was examined when KT-80 was administered to dogs which had been diagnosed as having natural canine parvovirus (CPV) infection. Only some cases in which the activity increased until 3 hr post-administration survived. These results suggest that rFeIFN-omega (KT-80) treatment enhanced the cellular immunity of normal dogs, and could exert significant therapeutic effects on only natural CPV infected dogs with induced continuous immunoenhancement.     

当归内酯对免疫抑制小鼠免疫功能的重建作用(英文)

探讨当归内酯(ASDL)对免疫抑制小鼠免疫功能的重构作用。通过小鼠腹腔注射环磷酰胺建立免疫抑制动物模型。采用免疫器官重量法和小鼠腹腔巨噬细胞吞噬鸡红细胞实验检测了ASDL对非特异性免疫功能的影响;用血清溶血素分光光度法检测了对体液免疫功能的作用;用MTT法进行了致分裂原诱导的小鼠脾淋巴细胞的增值反应实验,再用乳酸脱氢酶法测定了NK和CTL细胞活性,从而确定ASDL对小鼠细胞免疫功能的影响。结果表明:ASDL能够对免疫低下小鼠的非特性和特异性免疫功能起到一定的重构作用。但是这种效果并不是剂量依赖性的,20 mg/kg这个剂量的效果明显好于5和80 mg/kg这两个剂量。上述结果表明ASDL能够显著提高免疫低下小鼠的免疫功能。     

The activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase in rat tissues

The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was measured in 14 rat tissues and in subcellular fractions of rat liver by a sensitive fluorometric method, using cyclic NADP as substrate. CNP activity in brain (339 mumol/h/mg protein) was fourfold that of the sciatic nerve. The activities in tissues outside the nervous system ranged from a low of 0.42 mumol/h/mg protein in the unwashed red blood cell to a high of 9.96 in the spleen. The activity was highest in tissues containing cells with membranes capable of undergoing transformation and elaboration (spleen and thymus) and low in those in which the cell membranes are morphologically stable (muscle and red cell). The enzyme was found in all major liver subfractions, with the highest activities in the microsomal and nuclear fractions. Despite the large difference in the maximal velocities of CNP in brain and liver, the affinity of the liver enzyme for the substrate (km) was similar to that of brain enzyme. Brain CNP was stable over a 48-h postmortem period.     

Comparison of the biological properties of purified natural and recombinant human interleukin-2

We compared the biological properties of the purified recombinant human IL-2 derived from E. coli with those of purified natural IL-2. Both had almost the same specific in vitro activities on a weight basis to support long-term proliferation of IL-2 dependent human peripheral blood lymphocytes, a mouse killer T cell line, and a mouse natural killer cell line; induce killer cells in normal mouse spleen cells; and induce antibody forming cells in nude mouse spleen cells. No differences in these biological activities were found between two forms of natural IL-2 that were separable by reverse phase high performance liquid chromatography.     

Marker and function analysis of natural killer and alloreactive T cells during early stages of dimethylbenzanthracene carcinogenesis. The immunity system during the precancer period

The effect of the chemical carcinogen dimethylbenzanthracene (DMBA) on cellular immunity was studied at a 6-mg dose which induces adenocarcinomas and adenoacanthomas in more than 70% of BalB/c mice within 1 year after administration. DMBA caused a significant reduction of splenic natural killer (NK) activity and responsiveness to alloantigens in mixed lymphocyte reactions (MLR). These activities decreased soon after the carcinogen treatment and remained suppressed during the entire tumor induction period. There was a linear correlation between the reduction in NK activity and a selective decrease in the number of asialo GM1 positive cells in the spleen. However, cell sorting experiments using the flow cytometer have shown that the lytic activity per cell of asialo GM-1 positive cells in untreated mice and in DMBA-treated ones was similar. There was no correlation between the suppressed response of the T cells in MLR and the percentage of T cell subpopulations residing in the spleen of the DMBA-treated mice. The decrease in the number of NK cells and the reduced MLR activity in the spleen occurred simultaneously with a decrease in the potential of bone marrow precursor cells to reconstitute NK and MLR activity in the spleen of lethally irradiated mice. These results indicate that the carcinogen DMBA effects the immune system at various levels and either eliminates or inactivates precursor cells as well as mature lymphoid cells.     

Natural killer cell activity in mouse aggregation chimeras

The activity of natural killer (NK) cells in spleen against syngeneic and allogeneic tumor cells was studied by the use of tetraparental mouse chimeras. Chimeras were produced by aggregation of early embryos of histoincompatible mouse strains of “high” and “low” NK cell activity. NK activities of spleen cells were assayed in vitro by the ⁵¹Cr-release method. Coat color distribution and isozymal analysis (glucose-phosphate isomerase) of several lymphoid organs (thymus, lymph nodes, and bone marrow) revealed a predominant share of the “high”-NK-reactive genotype in the chimeras. However, the cellular NK activity against two target cell lines differing in their susceptibility to lysis was significantly lower in chimeras than in the “high”-reactive strain. Addition of “low”-NK spleen cells or of NH₄Cl-inactivated “high”-NK spleen cells to “high”-NK spleen cells inhibited their cytolytic activity. Possible mechanisms of the suppression of the cytolytic capacity of NK cells in chimeras are discussed.     

纳洛酮和电针改善创伤应激大鼠的免疫功能

本工作研究了中枢阿片肽系统在手术创伤介导大鼠免疫功能低下效应中的作用以及电针对创伤介导的免疫功能低下效应的影响。     

Enhanced immunostimulatory and antitumor activity of different derivatives of κ-carrageenan oligosaccharides from <Emphasis Type="Italic">Kappaphycus striatum</Emphasis>

Chemical modification of carbohydrates can lead to differences in their biological activities. We previously showed that κ-carrageenan oligosaccharides from Kappaphycus striatum have antitumor and immunomodulation effects on S180-bearing mice. In this study, we tested the hypothesis that different chemical modifications of carrageenan oligosaccharides enhance their activities. The mice inoculated with S180 cell suspension were treated p.o. with carrageenan oligosaccharides and their sulfated, acetylated, and phosphorylated derivatives (50, 100, and 200 μg g⁻¹) for 14 days. Transplantable tumor inhibition rate and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells, lymphocyte proliferation, the activity of natural killer cells, production of interleukin-2, and tumor necrosis factor-α were also analyzed. As expected, treatment with different κ-carrageenan oligosaccharides derivatives resulted in an increase in tumor inhibition rate and macrophage phagocytosis and cellular immunity, especially on spleen lymphocyte proliferation. The sulfated derivative at the dose 200 μg g⁻¹ per day showed the highest antitumor activity with the 54.12% tumor weight inhibition and elicited an increase in nature killer cells activity up to 76.1% on S180-bearing mice, which were both significantly higher than the unmodified oligosaccharides. It suggested that chemical modification (especially sulfation) of carrageenan oligosaccharides can enhance their antitumor effect and boost their antitumor immunity.     

Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retarets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8⁺ and CD4⁺ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V11, in particular CD8⁺ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.     

Glucocorticoid receptor mediated suppression of natural killer cell activity: identification of associated deacetylase and corepressor molecules

Physical and psychological stressors reduce natural killer cell function. This reduction in cellular function results from stress-induced release of glucocorticoids. Glucocorticoids act upon natural killer cells to deacetylate and transrepress immune response genes through epigenetic processes. However, other than the glucocorticoid receptor, the proteins that participate in this process are not well described in natural killer cells. The purpose of this study was to identify the proteins associated with the glucocorticoid receptor that are likely epigenetic participants in this process. Treatment of natural killer cells with the synthetic glucocorticoid, dexamethasone, produced a significant time dependent reduction in natural killer cell activity as early as 8h post treatment. This reduction in natural killer cell activity was preceded by nuclear localization of the glucocorticoid receptor with histone deacetylase 1 and the corepressor, SMRT. Other class I histone deacetylases were not associated with the glucocorticoid receptor nor was the corepressor NCoR. These results demonstrate histone deacetylase 1 and SMRT to associate with the ligand activated glucocorticoid receptor within the nuclei of natural killer cells and to be the likely participants in the histone deacetylation and transrepression that accompanies glucocorticoid mediated reductions in natural killer cell function.     

Selenium content and distribution in rat tissues irradiated with gamma rays

The effects of supplementation with selenous yeast and ionizing radiation on selenium (Se) content and distribution were evaluated in rat tissues (liver, kidney, spleen, heart, muscle, blood, front brain, hind brain, hypothalamus, pituitary, adrenal glands, testes, and hair). This study had 16 Se-supplemented (0.5 μg Se/d) and 16 placebo adult male Wistar rats. One half of the animals (eight Se-supplemented and eight placebos) were irradiated with a single dose of 4.2 Gy from a Co-60 source and sacrificed 7 d after irradiation along with nonirradiated animals and analyzed for Se content determination. The data obtained showed that selenous yeast supplementation increased Se levels in rat tissues (highest increases in hypothalamus, 161%; hind brain, 126%; spleen, 110%; and adrenal gland, 105%). Ionizing radiation induced significant changes in Se content and distribution (decrease in liver, blood, hair, femoral muscle, spleen, and hypothalamus; increase in kidney, testes, adrenal glands, and brain of placebo group). Supplementation with selenous yeast reduces changes in Se content and distribution after irradiation. It seems that the animal tissue susceptibility to oxidative damage may be correlated to their ability to retain Se in tissues.     

Hypothalamic control of the generation of mature natural killer lymphocytes in bone marrow and spleen of the mouse

Following previous work showing that electrothermocoagulation of the median region of the hypothalamus (MH) caused a marked and permanent decrease in the cytotoxicity of natural killer (NK) cells and in the number of large granular lymphocytes, a study was made of the effect of such lesions on the generation of NK cells in the bone marrow (BM) and spleen of C57BL/6 mice. Fresh spleen and BM cells from MH-lesioned and sham-operated mice were cultured with 40 U of recombinant interleukin-2 (rIL-2). NK activity was significantly higher in BM of lesioned mice, whereas spleen NK activity was greater in the sham-operated controls. NK cells matured by culture with rIL-2 were characterized by assay with fluorescent monoclonal antibodies and found to display the typical NK phenotype. These results show that the number of NK precursors is greater in BM of MH-lesioned mice and that their migration into other organs is probably partially impeded. It can also be concluded that intactness of both BM and the hypothalamus is essential for the physiological generation of NK cells.     

Effect of repeated oral administration of quinalphos to male goat (Capra hircus)

Quinalphos given in daily oral doses of 0.5 mg/kg for 110 days induced severe signs of organophosphorus poisoning in male goats. The inhibition of acetylcholinesterase activity in erythrocyte was highly significant. The activity of liver glutamic; oxaloacetic transaminase, glutamic; pyruvic.transaminase, alkaline phosphatase and protein indicated marked alteration. The haematological changes were however, relatively less significant with the exception of a very low count of red blood cells and white blood cells in the treated animals. Among the vital organs, only liver suggested mild cellular changes due to quinalphos intoxication. There was no significant pathological change in other organs of the treated animals. In animals observed after 15 and 30 days rest, the activity of acetylcholinesterase in red blood cells and haematological picture showed a fairly good recovery. This study suggests that although quinalphos in low concentrations did not produce discernible cellular changes, it induced highly significant enzymatic and haematological changes in the goat.     

Interstrain differences in cellular glucocorticoid reception in mice and the degree of suppression of normal killer activity during immobilization stress

The correlation between the level of stress-induced natural killer (NK) depression and glucocorticoid binding to specific spleen cell receptors and hormonal profile in inbred mouse strains (CBA, BALB/c, C57BL/c, A/Sn) has been investigated. Stable interstrain differences in stress-induced natural killer activity and glucocorticoid receptor binding (Bm and Kd) have been revealed. It was demonstrated that the mechanism of NK activity depression during stress consists in genetically determined potential sensitivity of lymphoid cells to physiological fluctuations of glucocorticoid levels. This made it possible to identify stress-resistant and stress-sensitive mouse strains.     

Identification of a chicken CLEC-2 homologue, an activating C-type lectin expressed by thrombocytes

Receptors on natural killer (NK) cells are classified as C-type lectins or as Ig-like molecules, and many of them are encoded by two genomic clusters designated natural killer gene complex (NKC) and leukocyte receptor complex, respectively. Here, we describe the analysis of an NKC-encoded chicken C-type lectin, previously annotated as homologue to CD94 and NKG2 and thus designated chicken CD94/NKG2. To further elucidate its potential function on NK cells, we produced a specific mab by immunizing with stably transfected HEK293 cells expressing this lectin. Staining of various chicken tissues revealed minimal reactivity with bursal, or thymus cells. In peripheral blood mononuclear cell and spleen, however, the mab reacted with virtually all thrombocytes, whereas most NK cells in organs such as embryonic spleen, lung and intestine were found to be negative. These findings indicate that the gene may not resemble CD94/NKG2, but rather a CLEC-2 homologue, a claim further supported by sequence features such as an additional extracellular cysteine residue and the presence of a cytoplasmic motif known as a hem immunoreceptor tyrosine-based activation motif, found in C-type lectins such as Dectin-1, CLEC-2, but not CD94/NKG2. The biochemical analyses demonstrated that CLEC-2 is present on the cell surface as heavily glycosylated homodimer, which upon mab crosslinking induced thrombocyte activation, as measured by CD107 expression. These analyses reveal that the chicken NKC may not encode NK cell receptor genes, in particular not CD94 or NKG2 genes, and identifies a chicken CLEC-2 homologue.     

Peyer's patch lymphocytes express natural cytotoxicity but not natural killer activity

The distribution of natural cytotoxic (NC) cells in the gut-associated lymphoid tissues (GALT) and in peripheric lymphoid organs was analyzed in comparison to that of natural killer (NK) cells. It was found that cells from the intestinal epithelium, mesenteric lymph nodes and spleen possess significant levels of NC and NK activity, whereas in thymus and popliteal lymph nodes both the natural activities are negligible. As previously shown for splenocytes, the NC activity of GALT cells is detectable in the 16-hour assays and not in the 4-hour assays. Interestingly, Peyer patches lymphocytes (PPL) possess extremely high NC activity but no NK activity. The NC activity of PPL is still high in NK-deficient mouse strains such as A/J and SJL/J. To further support the observation that the effector PPL are truly NC cells, it was shown that, as previously reported for spleen NC activity, overnight incubation at 37 degrees C of the lymphocytes only marginally affected the cytotoxicity of PPL, which could in turn be augmented by interleukin-3 (IL-3) containing supernatants. On the contrary, IL-2 could not increase NC or NK activity by PPL whilst augmenting NK activity of splenocytes. Thus, for the first time a cell population is identified which expresses only NC activity and not NK and which can be positively regulated only by IL-3.     

Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.     

Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating which shows that 2-arachidonoylglycerol plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of 2-arachidonoylglycerol on the motility of human natural killer cells. We found that 2-arachidonoylglycerol induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by 2-arachidonoylglycerol was abolished by treating the cells with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the 2-arachidonoylglycerol-induced migration. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with 2-arachidonoylglycerol abolished the migration induced by 2-arachidonoylglycerol. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, 2-arachidonoylglycerol, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.