Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes.

The yeast Saccharomyces cerevisiae contains two primary sequence subtypes of histone H2B (H2B1 and H2B2) and of H2A (H2A1 and H2A2). Mutants in each of the H2B subtypes have been used to show previously that yeast cells lacking one or the other, but not both, of the H2B proteins are viable. Because H2A protein interacts in the nucleosome with H2B, we wished to determine whether specific H2A subtypes must interact with specific H2B subtypes. We describe experiments in which frameshift mutations were introduced into both of the H2A genes in vitro and the mutant genes integrated into the yeast genome, replacing the wild-type H2A genes by a subsequent recombination. Using these mutant (hta1- and hta2-) strains we find that neither H2A gene has a unique essential function during any phase of the yeast life cycle, although strains homozygous for hta1- grow more slowly. However, one functional H2A gene is required for viability because cells mutant in both H2A genes arrest at spore germination prior to bud separation. By combining these H2A mutations with the H2B mutations obtained previously, we show that all combinations of H2A and H2B subtypes produce viable cells. From these genetic experiments and electrophoretic analysis of the histone proteins of these mutants we conclude that the H2A subtypes can associate interchangeably with the H2B subtypes.     

Expression of plasminogen activator as a marker of stimulation in tumor-associated macrophages

Tumor-associated macrophages (TAM) are a peculiar subpopulation of resident phagocytes with activities possibly important at the tumor host interface. Among these activities the production of proteases could obviously influence tumor cell detachment and migration from the primary. We have evaluated the expression of plasminogen activator (PA) in different types of murine tumors with differing immunogenic capacity. In TAM from all tumors studied the expression of PA was markedly greater than that of resident peritoneal macrophages. The fact that PA was similarly enhanced in peritoneal macrophages responding to a standard stimulation (thioglycolate) and in TAM suggests that the expression of PA is part of a more general cellular inflammatory response to injury.     

Identification of a functionally important sequence in the C terminus of the interferon-gamma receptor.

We have previously shown that the intracellular domain of the interferon-gamma (IFN-gamma) receptor plays an obligate role in receptor-mediated signal transduction. Moreover, we have specifically identified two regions within the human IFN-gamma receptor's intracellular domain required for functional activity: the membrane-proximal 48 amino acids required for both functional activity and receptor-mediated ligand internalization and the C-terminal 39 amino acids required exclusively for biologic response induction. Herein we report the identification of the 3 amino acids within the C-terminal region of the receptor that are obligatorily required for receptor function. By using a set of overlapping truncation mutants, the minimal functional sequence within the C-terminal region was localized to residues 434-444 (APTSFGYD-KPH). By mutating each individual residue within this sequence to alanine, three residues (Tyr-440, Asp-441, and His-444) were identified as being critical for IFN-gamma-dependent (i) upregulation of major histocompatibility complex class I proteins, (ii) activation of the IFN regulatory factor 1 gene, and (iii) stimulation of cells to produce nitric oxide. The more conservative Tyr-440-->Phe substitution also resulted in a nonfunctional receptor. Subsequent mutational analysis of all five of the IFN-gamma receptor's intracellular tyrosine residues revealed that Tyr-440 was the sole tyrosine required for receptor activity. These results thus identify a unique sequence in the IFN-gamma receptor that is required for initiation of IFN-gamma-dependent biologic responses and highlight the importance of the hydroxyl side chain of Tyr-440 in this process.     

Lysophosphatidylcholine induces urokinase-type plasminogen activator and its receptor in human macrophages partly through redox-sensitive pathway

Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) have been shown to be expressed in macrophages in atherosclerotic arterial walls, but the regulatory mechanisms of their expression remain unclear. The present study was performed to examine the effects of lysophosphatidylcholine (lysoPC), an important atherogenic lipid, on the expression of uPA and uPAR in human monocyte-derived macrophages. LysoPC upregulated the mRNA expression of uPA and uPAR, and it increased the protein expression of uPA in the culture medium and bound to the cell surface and of uPAR in the particulate fraction of the cells. LysoPC significantly increased the binding of the amino-terminal fragment of uPA to the treated cells and the cell-associated plasminogen activator activity. LysoPC stimulated superoxide anion production and increased intracellular oxidant levels in the cells. The combined incubation with reduced glutathione diethyl ester or N-acetylcysteine, antioxidants, suppressed the upregulation of uPA and uPAR mRNA and the increase in plasminogen activator activity by lysoPC. uPA and uPAR mRNA expression was also induced by the incubation with xanthine and xanthine oxidase, a superoxide anion-generating system. The results suggest that lysoPC increased the expression of uPA and uPAR and their functional activities in human monocyte-derived macrophages, at least in part through a redox-sensitive mechanism. This coordinate increase in the expression of uPA and uPAR in human macrophages by lysoPC could play an important role in plaque formation and disruption, arterial remodeling, and angiogenesis in atherosclerotic arterial walls.     

Nicotinic acetylcholine receptor: evidence for a functionally distinct receptor on human lymphocytes.

The presence of three distinct cholinergic receptors on human lymphocytes was suggested by the effects of carbamoylcholine on lymphocyte proliferation in vitro. The cells responded to both 0.1 nM and 1 microM carbamoylcholine by increased proliferation which was blocked by the muscarinic antagonist atropine. This effect occurred in both mitogen-stimulated cells (maximum effect at 24 hr) and nonstimulated cells (maximum effect at 72 hr). In contrast, 1--10 nM carbamoylcholine produced diminished in vitro proliferation, an effect which was blocked by the nicotinic antagonists alpha-bungarotoxin and d-tubocurarine.     

Histone H2A variant synthesis in aging human diploid cells

The synthesis of the various classes of core particle histones was determined in human diploid fibroblast-like cells of different in vitro ages as they were stimulated to enter the cell cycle. Histone H2A synthesis in older populations was lower during G1 but was similar to younger cells at G0/G1 and S phases. The reduced synthesis during G1 was primarily the result of a decline in the synthesis of the H2A.1/.2 component.     

Histone variants of H2A and H3 families are regulated during in vitro aging in the same manner as during differentiation

In a previous communication, we showed that the H2A.1/H2A.2 histone variant ratio decreases in a linear manner during the in vitro aging of human diploid fibroblasts. This ratio is known to decrease in the same manner in progressive stages of development and in the process of differentiation, and is thus considered to be a biochemical marker for differentiation. A detailed analysis of the synthesis of H2A and H3 histone variants as a function of cumulative population doublings in the same in vitro cell system is presented in this study. Quantitative analysis of these variants in the G0 phase, synchronized fibroblasts has shown that their relative amount in chromatin, as well as their biosynthesis rate, change during in vitro aging of human diploid fibroblasts, revealing both up-and down-regulation of certain variants as a function of cumulative population doublings. Furthermore, we show by morphometric studies employing the seven distinct fibroblast morphotypes, as described by the Bayreuther classification, that this regulation is attributable to the replicative sub-populations. These results reveal that histone variants of the H2A and H3 families are regulated during in vitro aging in the same manner as that during differentiation.     

Gene transfers of kallikrein, bradykinin receptor B2 and a mutated B2 receptor stimulate neoangiogenesis in peripheral ischemia

In this work, we evaluated the angiogenic effect of the gene transfer of human tissue kallikrein (TK), bradykinin B2 receptor (B2R) and a mutated form (RB2m) in a rabbit peripheral model of ischaemia. We studied the effects of the transfection of each of these factors and the effects of their co-transfection. In New Zealand anesthetised rabbits we first induced an ischaemia of the left posterior leg by ligation-excision of the superficial femoral artery and its collaterals. Seven days later, we performed i.m. injections in the ischemic tight with transfection solutions containing either the control (pcDNA3 empty backbone) or the pcDNA3-TK, the pcDNA3-TK and the pcDNA3-B2R, the pcDNA3-TK and the pcDNA3-B2Rm. Twenty eight days later, the therapeutic effect was evaluated using ultrasonographic debitmetry of the common iliac artery, perfusion index (PI) = ischemic leg blood flow /non ischemic leg blood flow (%) and capillaries measurements i.e. capillary density: number of vessels/mm2 and the ratio of vessels/muscular fiber, in the adductors and gastrocnemian muscles. The PI was increased in each treated group vs control (32.61 +/- 5.2%), pcDNA3-TK: 59.72 +/- 2.33%; p = 0.001; pcDNA3-RB2: 55.25 +/- 2.29%; p = 0.008; pcDNA3-TK + pcDNA3-RB2: 84.77 +/- 3.15%; p < 0.001; pcDNA3-TK + pcDNA3-RB2m: 103.25 +/- 4.9%; p < 0.001. The capillary density and the vessel/muscular fiber ratio increased in a parallel with the hemodynamic in the ischemic adductors (pcDNA3-TK + pcDNA3-B2Rm > pcDNA3-TK + pcDNA3-RB2 > pcDNA3-TK = pcDNA3-B2R; p < 0.001). There was no angiogenic effect measurable neither in the non ischemic adductors (right) nor in the gastrocnemian muscles. In rabbit peripheral ischaemia, the cotransfection of TK and B2R increases the arterial flow in the treated leg and potentiates the neoangiogenesis. Cotransfection of the B2Rm cDNA enhanced the synergic effect of this therapeutic strategy.     

Soluble urokinase plasminogen activator receptor as a prognostic marker in men participating in prostate cancer screening

Abstract. Kjellman A, Akre O, Gustafsson O, Høyer‐Hansen G, Lilja H, Norming U, Piironen T, Törnblom M (Department of Clinical Science, Intervention, and Technology; Clinical Epidemiology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Copenhagen, Denmark; Departments of Clinical Laboratories, Urology and Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden; and Syrinx Bioanalytics Oy, Turku, Finland; formerly at Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Copenhagen, Denmark). Soluble urokinase plasminogen activator receptor as a prognostic marker in men participating in prostate cancer screening. J Intern Med 2010; 269 : 299–305. Background. The urokinase plasminogen activator (uPA) system is involved in tissue remodelling processes and is up‐regulated in many types of malignancies. We investigated whether serum levels of different forms of soluble uPA receptor (suPAR) are associated with survival and in particular with prostate cancer and cardiovascular disease mortality. Methods. Using time‐resolved fluorescence immunoassays, we measured intact suPAR [suPAR(I‐III)] and intact plus cleaved suPAR [suPAR(I‐III) + suPAR(II‐III)] and thus calculated the amount of suPAR(II‐III) in serum samples from 375 men participating in a prostate cancer screening trial. A total of 312 men were free of prostate cancer and 63 men had prostate cancer diagnosed at the time of screening. The cohort was followed for 15 years. We assessed survival using Kaplan–Meier estimation and Cox proportional hazards regression. Results. The mean age at blood sampling was 64 years. In total, 152 men died during follow‐up. SuPAR(I‐III) and suPAR(II‐III) were significantly positively associated with mortality (P = 0.001 and P < 0.0001, respectively). In a Cox regression model adjusting for age and prostate cancer status, an increase in suPAR(I‐III) and suPAR(II‐III) by 1‐unit (ln‐scale) was associated with a hazard ratio (HR) of 2.26 [95% confidence interval (CI) 1.17–4.35] and 2.53 (95% CI 1.56–4.10), respectively. There was a trend towards an increased risk of death from prostate cancer in screening‐detected prostate cancer patients with increased values of either suPAR form. However, this difference was not significant and the association disappeared after adjusting for age, tumour stage, tumour grade and prostate‐specific antigen. Being in the highest quartile of any of the suPAR forms was associated with a highly significant increased risk of cardiovascular death, with HR adjusted for age of 3.27 (95% CI 1.38–7.73) for suPAR(I‐III) quartile 4 versus quartile 1. Conclusions. High concentrations of serum suPAR(I‐III) and suPAR(II‐III) were associated with poor overall survival. The association was particularly strong for death from cardiovascular disease. No similar association was found for prostate cancer after adjustment for other prognostic factors.     

Microvascular endothelial cells express a phosphatidylserine receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes

Phosphatidylserine (PS)-positive erythrocytes adhere to endothelium and subendothelial matrix components. While thrombospondin mediates these inter-actions, it is unknown whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control HbAA erythrocytes, we demonstrate that PS-positive erythrocytes adhered to human lung microendothelial cells in the absence of plasma ligands, that this adhesion was enhanced following endothelial activation with IL-1alpha, TNF-alpha, LPS, hypoxia, and heme, and that this adhesive interaction was selective to erythrocyte PS. We next explored whether microendothelial cells express an adhesion receptor that recognizes cell surface-expressed PS (PSR) similar to that expressed on activated macrophages. We demonstrate constitutive expression of both PSR mRNA and protein that were up-regulated in a time-dependent manner following endothelial activation. While minimal PSR expression was noted on unstimulated cells, endothelial activation up-regulated PSR surface expression. In antibody-blocking studies, using PS-positive erythrocytes generated either artificially via ionophore treatment of control erythrocytes or from patients with sickle cell disease, we demonstrate that PSR was functional, supporting PS-mediated erythrocyte adhesion to activated endothelium. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the microendothelium that is up-regulated by such pathologically relevant agonists as hypoxia, cytokines, and heme.     

Soluble urokinase plasminogen activator receptor suPAR as a marker for inflammation in pediatric inflammatory bowel disease

Abstract

Objective. In inflammatory bowel disease (IBD), more means to monitor early therapeutic response are needed. In pediatric IBD, blood inflammatory markers erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) may be low in 10 to 20% of patients with severe disease. Recently, soluble urokinase plasminogen activator receptor (suPAR) was described as a potential blood inflammatory marker in adult IBD. Methods. We tested the performance of suPAR by the start of therapy with glucocorticoids (n = 19) or TNF-α-antagonist (n = 16) in pediatric IBD (Crohn's disease n = 19, ulcerative colitis (UC) n = 16). Results. The levels of suPAR were low in both patient groups studied. There was no difference in the values regarding the presence of Crohn's disease or ulcerative colitis. Thus, all analyses were performed on the entire sample set. Glucocorticoid therapy, however, resulted in a significant decline in suPAR levels from a median of 3.06 to 2.54 ng/ml (p < 0.01). In contrast, TNF-α-antagonist had no effect. The suPAR levels did not associate with ESR or CRP or fecal calprotectin (FC). Conclusions. In pediatric IBD, the suPAR levels in blood are low and do not reflect the level of intestinal inflammation assessed with FC. The introduction of corticoids, however, results in a decline of suPAR levels in blood but not reflect therapeutic response to TNF-α-antagonist. Thus, suPAR is of limited value in assessing systemic inflammatory responses in pediatric IBD.