Age-related differences in the induction of 2-5A synthetase and 2-5A dependent binding protein activities by interferon in guinea pig peritoneal macrophages

2-5A synthetase and binding protein activities in peritoneal macrophages have been compared between young (6 month) and old (22-24 month) guinea pigs. Enzyme activities are lower in aged animals with a 17% and a 31% reduction in synthetase and binding protein activities, respectively. In addition, the response to the addition of mouse fibroblast interferon by macrophages from these two age groups is also substantially different. Whereas addition of interferon to young guinea pig macrophages elicits a 3.8- and a 1.7-fold increase in the synthetase and binding protein activities, only a marginal elevation in these two enzyme activities is found with interferon-treated old guinea pig macrophages. Analysis by thin layer chromatography demonstrates a marked difference in the relative distribution of the various oligomeric forms of 2-5A synthesized by young or old guinea pig macrophages. The binding protein in old animals appears to be significantly more thermolabile than the corresponding activity from young animals. The altered response to interferon and the difference in enzymatic properties in aged animals may represent part of the mechanisms involved in the progressive loss of the adaptative ability of an organism to environmental changes during senescence.     

Demonstration of 2-5A synthetase in human circulating mononuclear cells after oral administration of an interferon inducer isolated from a fraction of Escherichia coli and Klebsiella pneumoniae

Evaluation of the activity of an enzyme activated by interferon (2'-5' oligo-isoadenylate synthetase or 2-5A synthetase) has been used to determine, in man, the interferon inducing capacity of a fraction isolated from Escherichia coli and Klebsiella pneumoniae, SL04, an immunomodulating agent administrated per os. An increase of the activity of this enzyme has been shown in four out of five volunteers following a single oral administration of 100 mg of SL04. This activation demonstrated in humans confirms the pharmacological results of the interferon induction obtained with SL04 in vivo in mice and in vitro in human cell cultures.     

Substrates for the cell surface protein kinase in blood: a calf serum protein and its precursor in plasma

A protein in calf serum with molecular mass of 125,000 is selectively phosphorylated by the surface kinase activity of intact tissue culture cells and erythrocytes. The protein, termed pp125, is phosphorylated at serine and threonine residues to a ratio of greater than 1 mol P/mol. The pp125 is an acidic protein (pI 4.4) which also serves as substrate for purified phosvitin/casein kinases but not for cyclic AMP-dependent protein kinases. About 80-fold purification of pp125 was achieved by ion exchange and affinity chromatography. Gel filtration under non-reducing conditions showed that pp125 is part of a complex (Mr 535,000). The pp125 obviously originates from a large plasma protein: the incubation of calf plasma with intact cells in the presence of [gamma-32P]ATP resulted in the labeling of a protein with Mr greater than 300,000 (pp greater than 300). The relationship between pp greater than 300 in plasma and pp125 in serum was demonstrated by cyanogen bromide peptide patterns, and the use of specific anti-serum raised against pp125. Furthermore, it was shown that pp125 is derived from pp greater than 300 during blood clotting.     

Decreased Epstein-Barr virus-induced transformation, and elevated 2-5A synthetase and RNase L activity in peripheral blood mononuclear cells from patients treated with recombinant interferon alfa 2b

Patients with cutaneous T-cell lymphoma (CTCL) were treated with recombinant alfa 2b interferon (rIFN alfa 2b) by intramuscular injection. Therapy-induced changes in Epstein-Barr virus (EBV) induced transformation of patient peripheral blood lymphocytes, 2',5' oligoadenylate (2-5A) synthetase levels and RNase L activation in peripheral blood mononuclear cells were monitored. Inhibition of EBV-induced transformation and elevation of 2-5A synthetase levels correlated with increased activation of RNase L, which provides evidence that intramuscular administration of rIFN alfa 2b induces a sustained anti-EBV state in CTCL patient peripheral blood mononuclear cells which can be detected in vitro.     

A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization

Rat brain tryptophan 5-monooxygenase was activated by incubation with ATP, Mg2+, calmodulin, and micromolar concentrations of Ca2+. The activating activity was resolved into two distinct peaks upon gel filtration on Sepharose CL-6B: one, Ca2+-, calmodulin-dependent protein kinase, and the other, a heat-labile activator protein. The activator protein was purified to apparent homogeneity from rat brain by a procedure involving calmodulin-Sepharose 4B, Sephadex G-150, and phenyl-Sepharose CL-4B column chromatography. The molecular weight of the activator protein was determined to be 70,000 by sedimentation equilibrium and by gel filtration on Sephadex G-150. The protein gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 35,000, indicating that the protein might be composed of two identical subunits. Analysis of cross-linked activator protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also suggested that the protein might be a dimer of identical subunits. Some other molecular properties of the activator protein were: sedimentation coefficient, 4.3 S; Stokes radius, 3.6 nm; diffusion coefficient, 6.0 x 10(-7) cm2/s; frictional ratio, 1.32; and partial specific volume, 0.73 cm3/g. The activator protein activated tyrosine 5-monooxygenase as well as tryptophan 5-monooxygenase in the presence of ATP, Mg2+, Ca2+, calmodulin, and Ca2+-, calmodulin-dependent protein kinase.     

Ligand-stimulated downregulation of the alpha interferon receptor: role of protein kinase D2

Alpha interferon (IFN-α) controls homeostasis of hematopoietic stem cells, regulates antiviral resistance, inhibits angiogenesis, and suppresses tumor growth. This cytokine is often used to treat cancers and chronic viral infections. The extent of cellular responses to IFN-α is limited by the IFN-induced ubiquitination and degradation of the IFN-α/β receptor chain 1 (IFNAR1) chain of the cognate receptor. IFNAR1 ubiquitination is facilitated by the βTrcp E3 ubiquitin ligase that is recruited to IFNAR1 upon its degron phosphorylation, which is induced by the ligand. Here we report identification of protein kinase D2 (PKD2) as a kinase that mediates the ligand-inducible phosphorylation of IFNAR1 degron and enables binding of βTrcp to the receptor. Treatment of cells with IFN-α induces catalytic activity of PKD2 and stimulates its interaction with IFNAR1. Expression and kinase activity of PKD2 are required for the ligand-inducible stimulation of IFNAR1 ubiquitination and endocytosis and for accelerated proteolytic turnover of IFNAR1. Furthermore, inhibition or knockdown of PKD2 robustly augments intracellular signaling induced by IFN-α and increases the efficacy of its antiviral effects. The mechanisms of the ligand-inducible elimination of IFNAR1 are discussed, along with the potential medical significance of this regulation.     

Expressed 2-5A synthetase genes and pseudogenes in the marine sponge Geodia barretti

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. In this study, we sequenced alleles of the BoLA class II loci, BoLA-DRB3 and BoLA-DQA1, from 650 Japanese cattle from six herds [three herds (507 animals) of Japanese Black cattle and three herds (143 animals) of Holstein cattle] using polymerase chain reaction-sequence-based typing (PCR-SBT) methods. We identified 26 previously reported distinct DRB3 alleles in the two populations: 22 in Japanese Black and 17 in Holstein. The number of DRB3 alleles detected in each herd ranged from 9 to 20. Next, we identified 15 previously reported distinct DQA1 alleles: 13 in Japanese Black and 10 in Holstein. The number of alleles in each herd ranged from 6 to 10. Thus, allelic divergence is significantly greater for DRB3 than for DQA1. A population tree on the basis of the frequencies of the DRB3 and DQA1 alleles showed that, although the genetic distance differed significantly between the two cattle breeds, it was closely related within the three herds of each breed. In addition, Wu-Kabat variability analysis indicated that the DRB3 gene was more polymorphic than the DQA1 gene in both breeds and in all herds, and that the majority of the hypervariable positions within both loci corresponded to pocket-forming residues. The DRB3 and DQA1 heterozygosity for both breeds within each herd were calculated based on the Hardy-Weinberg equilibrium. Only one Japanese Black herd showed a significant difference between the expected and observed heterozygosity at both loci. This is the first report presenting a detailed study of the allelic distribution of BoLA-DRB3 and -DQA1 genes in Japanese Black and Holstein cattle from different farms in Japan. These results may help to develop improved livestock breeding strategies in the future.     

Determination of serotonin in whole blood, platelet-rich plasma, platelet-poor plasma and plasma ultrafiltrate

The important biogenic amine, serotonin (5HT), was determined in whole blood, platelet-rich plasma (PRP), platelet-poor plasma (PPP), and plasma ultrafiltrate after simple deproteinization. Following ion-pair chromatography on standard or narrow-bore reverse-phase columns, 5HT and the internal standard (N-methylserotonin-NMS) were detected by fluorometry with absolute detection limits of 2-4 pg. Levels obtained for whole blood and PRP were in agreement with previous methods. However, mean (+/- SD) values obtained for platelet-poor plasma (PPP) of 578 +/- 277 pg/ml (N = 7) were approximately 3-fold lower than the lowest previous reports. We also report the first determination of 5HT in plasma ultrafiltrate, having observed mean levels of 387 +/- 222 pg/ml (N = 7).     

Evaluation of 2,5-oligoadenylate synthetase and protein kinase activities in clinical studies of larifan and recombinant interferon alpha in volunteers

The activity of the interferon-dependent enzymes: 2',5'-oligoadenylate synthetase and protein kinase was determined in blood specimens of volunteers in the clinical trials on reaferon (recombinant alpha 2-interferon) and larifan (replicate RNA of phage f2). It was shown that the preparations increased the activity of 2',5'-oligoadenylate synthetase in the lymphocytes and protein kinase in the plasma of 60 to 70 per cent of the volunteers. The increase in the 2',5'-oligoadenylate synthetase activity did not always correlate with the increase in the interferon content in the serum and was sometimes observed in the absence of the interferon. Marked individual variations in the activity of the enzymes were detected in the volunteers before and after administration of the preparations. The plasma kinase activated by reaferon and larifan phosphorylated proteins with molecular weights of 72 and 30 kD and histones. The effect of reaferon and larifan on the lymphocyte protein kinase activity was determined for the first time. There was a decrease in the enzyme activity under the effect of reaferon which increased after its repeated injections. Unlike the effect of larifan, the inhibitory effect of reaferon was transient. Afterwards, it appeared to be accompanied by a significant increase in the activity of protein kinase in 70 per cent of the volunteers. The dynamics of the changes in the activity of the plasma and lymphocyte protein kinases did not coincide.     

2',5'-Oligoadenylate synthetase and interferon in peripheral blood after rubella, measles, or mumps live virus vaccine

The temporal activation of the human interferon system by infection with virus was studied by serial measurements of both interferon in serum and activity of 2',5'-oligo adenylate synthetase in peripheral mononuclear leukocytes. A frequency distribution of baseline values of synthetase was established for normal individuals. Following subcutaneous inoculation of rubella vaccine virus, serum interferon rose briefly with a peak on Day 14. The peak concentration of synthetase also occurred on Day 14 but remained elevated for greater than 1 week. After measles virus, serum interferon did not rise above baseline, but synthetase peaked on Day 14 and remained elevated. Subcutaneous inoculation of mumps vaccine virus was associated with a brief period of elevation of the synthetase and no interferon in the serum. Thus, the determination of synthetase levels in tissue may be useful in some situations to reflect a small or transient elevation of endogenous interferon.     

Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM H+ -ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM H+ -ATPase AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM H+ -ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM H+ -ATPase regulation.     

Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis

The relative abundance and physiological role of 5alpha-reductase (5alphaR) isoforms in rat testis, in particular 5alpha-reductase Type 2 (5alphaR2) are poorly understood. Investigation of 5alphaR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5alpha-reductase Type 1 (5alphaR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5alphaR assays measured the conversion of 3[H]-testosterone to 5alpha-reduced metabolites (dihydrotestosterone+3alpha-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5alphaR1 activity at pH 5.0, the amount of 5alphaR1 activity at pH 5.0 was determined by measuring recombinant rat 5alphaR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5alphaR1 was determined to be 12.4+/-1.4% (mean+/-S.D., n=14). The 5alphaR2 assay was validated by determining recombinant rat 5alphaR2 activity in the presence of recombinant rat 5alphaR1 activity in COS cells. A 99.3+/-14.7% recovery of 5alphaR2 activity was obtained when comparing 5alphaR2 activity recovered versus activity added. 5alphaR1 and 5alphaR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50-100-fold) over this age range, with 5alphaR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5alphaR2 activity in the presence of high concentrations of 5alphaR1 was developed and is applicable in the measurement of 5alphaR2 activity in rat testis.     

Activators of protein kinase C and 5-azacytidine induce IL-2 receptor expression on human T lymphocytes

Resting human T lymphocytes do not express receptors for interleukin-2, but expression is rapidly induced by exposure to PHA. After maximal expression 2-3 days after stimulation, a progressive decline in receptor number is observed. Receptor expression can be augmented by reexposure to PHA. In this study we show that activators of protein kinase C including phorbol diester, phospholipase C, and the diacylglycerol congener diC8 also increase IL-2 receptor expression. Moreover, 5-azacytidine, which inhibits cytosine methyltransferase, and hydroxyurea, which inhibits ribonucleotide reductase, also increased receptor number. These studies demonstrate that IL-2 receptor expression can be altered in vitro, and that IL-2 receptor number, in combination with IL-2 secretion, may contribute to the regulation of IL-2-dependent immune responses.     

Minimal peripheral blood cells carrying clonal markers of B cell disorders: evidence for monoclonality of circulating lymphocytes in patients with multiple myeloma

Peripheral blood lymphocytes (PBL) of 20 patients with multiple myeloma (MM) were assayed for clonality by Southern blot and cell surface marker analysis. Eight samples showed monoclonal origin of circulating lymphocytes by demonstrating rearrangements of the heavy chain immunoglobulin gene (IgH). In selected experiments, comparison of IgH rearrangements of bone marrow plasma cells and peripheral blood-derived mononuclear cells, highly enriched for B lymphocytes, proved to be identical. However, monoclonal circulating cells could not be detected in samples with rearranged IgH genes by surface marker phenotyping using one-color immunofluorescence analysis and a panel of monoclonal and polyclonal antibodies to various B lineage-associated antigens. These results indicate that in a substantial proportion of MM, monoclonal growth involves circulating B lymphocytes and underscores the clinical usefulness of Southern analysis of IgH gene rearrangements for monitoring this disease.