Free radical-producing myeloid-derived regulatory cells: potent activators and suppressors of lung inflammation and airway hyperresponsiveness.

Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of inducible nitric oxide synthase (iNOS), arginase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of their infiltration into the lung. The nitric oxide-producing cells were Ly-6C(+)Ly-6G(-) and they downmodulated T-cell activation, recruited T(reg) cells, and dramatically downregulated antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C(-)Ly-6G(+) and they expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a superoxide-dependent fashion. A smaller population of Ly-6C(+)Ly-6G(+) cells also suppressed T-cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells represent important targets for asthma therapy.

Cell signalling by oxidized lipids and the role of reactive oxygen species in the endothelium.

The controlled formation of ROS (reactive oxygen species) and RNS (reactive nitrogen species) is now known to be critical in cellular redox signalling. As with the more familiar phosphorylation-dependent signal transduction pathways, control of protein function is mediated by the post-translational modification at specific amino acid residues, notably thiols. Two important classes of oxidant-derived signalling molecules are the lipid oxidation products, including those with electrophilic reactive centres, and decomposition products such as lysoPC (lysophosphatidylcholine). The mechanisms can be direct in the case of electrophiles, as they can modify signalling proteins by post-translational modification of thiols. In the case of lysoPC, it appears that secondary generation of ROS/RNS, dependent on intracellular calcium fluxes, can cause the secondary induction of H2O2 in the cell. In either case, the intracellular source of ROS/RNS has not been defined. In this respect, the mitochondrion is particularly interesting since it is now becoming apparent that the formation of superoxide from the respiratory chain can play an important role in cell signalling, and oxidized lipids can stimulate ROS formation from an undefined source. In this short overview, we describe recent experiments that suggest that the cell signalling mediated by lipid oxidation products involves their interaction with mitochondria. The implications of these results for our understanding of adaptation and the response to stress in cardiovascular disease are discussed.

Oxidative stress and heat shock stimulate RGS2 expression in 1321N1 astrocytoma cells.

RGS2, a regulators of G-protein signaling family member, regulates G-protein signaling and is itself controlled in part by regulated expression. We tested if cell stress regulates RGS2 expression in human astrocytoma 1321N1 cells. Treatment with H2O2 increased RGS2 mRNA levels time- and concentration-dependently, with 200 microM H2O2 causing an approximately eightfold increase after 2 h. Peroxynitrite and heat shock also increased RGS2 mRNA levels. H2O2-induced RGS2 expression was negatively regulated by phosphoinositide-3-kinase and extracellular signal-regulated kinases. H2O2 also concentration-dependently increased RGS2 protein levels, and the RGS2 appeared to be predominantly in the nucleus. These results demonstrate that RGS2 expression is up-regulated by cell stress.

RGS2: regulation of expression and nuclear localization.

RGS2, a Regulators of G-protein Signaling family member, regulates signaling activities of G-proteins, and RGS2 itself is controlled in part by regulation of its expression. This investigation extended previous studies of the regulation of RGS2 expression by examining the effects of stress, differentiation, and signaling activities on RGS2 mRNA level in human neuroblastoma SH-SY5Y cells. Cell stress induced by heat shock rapidly and transiently increased RGS2 mRNA levels, whereas differentiation to a neuronal phenotype reduced basal RGS2 mRNA levels by 50%. RGS2 mRNA levels were increased in differentiated cells by heat shock, carbachol, and activation of protein kinase C. After transient transfection of GFP-tagged RGS2, a predominant nuclear localization was observed by confocal microscopy. Thus, RGS2 expression is regulated by stress and differentiation, as well as by second messenger signaling, and transfected GFP-RGS2 is predominantly nuclear.

Second messengers regulate RGS2 expression which is targeted to the nucleus.

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.

[Analysis of newborns' health status in twin pregnancies according to the duration of pregnancy and its delivery based on the materials of Reproduction and Obstetrics Clinic in 1995-1999]. / Analiza stanu noworodków w ciazach blizniaczych w zaleznosci od czasu trwania ciazy i sposobu jej rozwiazania w materiale Kliniki Rozrodczosci i Poloznictwa we Wroclawiu w latach 1995-1999.

On the ground of the analysis of 79 twin pregnancies, the valuation of newborns' condition in Apgar's scale, according to the delivery means, has been executed. The lack of differences between the mean values of the scale has been ascertained. There was no indication of any difference between the condition of the twin I and II. Furthermore, there has been executed a detailed comparative analysis of the newborns' condition in the four periods of the pregnancy duration: 23-27, 28-32, 33-37 and 38-42 weeks of gestation. It has been ascertained that the newborns delivered through the abdominal delivery were in better condition than those born in the spontaneous delivery, in the 28-32 weeks of gestation period. The use of rather intraspinal rather than general anesthesia in the 33-37 weeks period gave better results by improving the newborns' condition. Moreover, there has been stated a similar condition of the newborns delivered through either spontaneous or abdominal delivery with intraspinal anesthesia in the periods of 33-37 and 38-42 weeks of gestation.

[Analysis of twin pregnancies on the ground of women hospitalized in the Department of Reproduction and Obstetrics Medical University in Wroclaw in 1995-1999]. / Analiza ciaz blizniaczych w oparciu o material Kliniki Rozrodczosci i Poloznictwa AM we Wroclawiu w latach 1995-1999.

The aim of this study was the general valuation of the course and the delivery means of the twin pregnancies. The research material composed of 83 from among 5540 pregnant women hospitalized in the Department of Reproduction and Obstetrics Medical University of Wroclaw in the years 1995-1999. The mean body mass values and the condition of the newborns have been analyzed on the ground of Apgar's scale, according to the date of delivery. In the period between 23 and 42 week of pregnancy a very high correlation between fetus' body mass and a high correlation between Apgar's scale and the pregnancy's duration has been ascertained. These values have also been estimated in particular periods: 23-27, 28-32, 33-37 and 38-42 weeks of gestation. Statistical analysis didn't indicate any difference between the mean values of Apgar's scale of the newborns from the periods of 33-37 and 38-42 weeks of gestation. There was no evidence of differences either in Apgar's scale values or in the twins' I and II body masses, as well in the whole examined group as in particular periods.

[Meaning of opening form of internal cervical opening in prognosis of cervical insufficiency]. / Znaczenie ksztaltu rozwarcia ujscia wewnetrznego kanalu szyjki macicy w rokowaniu w niewydolnosci ciesniowo-szyjkowej.

The introduction of the ultrasonographical examinations into the obstetrical diagnostics has created further possibilities in the process of recognition of the isthmico-cervical insufficiency. The qualification of the opening forms of the internal cervical os, described as Y, V and U forms, is possible with the utilization of transperineal and, especially, transvaginal sonography. The aim of this study was the valuation of the ultrasonographical examinations' usefulness in the diagnostics of the isthmico-cervical insufficiency and the estimation of the opening form of the internal cervical os as a prognostic factor in the pregnancy course. 265 cases of pregnant women hospitalized in Department of Fertility and Obstetrics Medical University of Wroclaw have been analyzed. On the ground of the executed examinations it has been ascertained that the ultrasonographical examination, considering the valuation of the opening form of the internal cervical os, is essential in the diagnostics of the isthmico-cervical insufficiency. Furthermore, it has been proved that in the aspect of the obtainment of the full-term pregnancy, the worst-prognosing form of the internal cervical os is the U form and the statement of a U or V formed opening confirms the necessity of the circular suture of the cervix.

[Fibronectin level in amniotic fluid as an index of unavoidable labor]. / Stezenie fibronektyny w plynie owodniowym jako wykladnik nieuchronnego porodu.

During uterus contractions detaching of amino-chorionic layer from uterus wall occurs and released fibronectin penetrates into amniotic fluid. The aim of this study was to estimate in a quantity mode the presence of fibronectin in amniotic fluid and to find the dependence between the fibronectin level in amniotic fluid and the period of time from collecting the sample to the labor. We wanted also to find the dependence between fibronectin level in amniotic fluid and duration of pregnancy, preterm rupture of amniotic membranes, patients' age, parity and number of deliveries. We analysed 86 pregnant women where we estimated the fibronectin level in specimens of amniotic fluid. During carrying out the experiment we noted that fibronectin is present in amniotic fluid and can be identified in a quantity mode. We have proved dependence between fibronectin level in amniotic fluid and the period of time from collecting the sample, up to the delivery. Fibronectin level in amniotic fluid in pregnancies uncomplicated with premature delivery was on the average 350 mg/ml. Increase of fibronectin in amniotic fluid above 700 mg/ml points at detaching of amino-chorionic layer and the occurrence of unavoidable preterm labor at the time no longer than 24 hours. Fibronectin level in amniotic fluid doesn't depend of pregnancy duration, preterm rupture of amniotic membranes.

Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus.

The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.