Determination of ultra-low concentrations of gaseous 14C-bearing hydrocarbons produced during corrosion of irradiated steel using accelerator mass spectrometry.

Compound-specific radiocarbon analysis (CSRA) was developed to identify and quantify gaseous 14C-bearing carbon compounds at the pico- to femtomolar concentration range and employed in a corrosion experiment with small specimens of irradiated steel. The approach is based on gas chromatographic separation of single 14C-bearing carbon compounds, their oxidation to 14CO2, sampling with a custom-made fraction collector and quantification by accelerator mass spectrometry (AMS). In addition to CSRA, a method allowing the quantification of the total 14C content of the gas phase was developed and tested. After validation of the two set-ups with standards, the gaseous 14C-bearing carbon compounds produced during alkaline anoxic corrosion of irradiated steel were quantified. Small hydrocarbons (HCs) like methane (14CH4) and ethane (14C2H6) were the only 14C-bearing compounds identified in the gas phase above the detection limit. 14CH4 was the main species (on average 5.4 × 10-14 mol L-1 gas) and contributed >90% to the total 14C content, whereas the concentration of 14C2H6 was much lower (7.9 × 10-16 mol L-1 gas). To our knowledge, this is the first study reporting CSRA of gaseous 14C-bearing HCs produced during anoxic corrosion of irradiated metallic radioactive waste at ultra-low concentrations.

Analysis of 14C-bearing compounds released by the corrosion of irradiated steel using accelerator mass spectrometry.

The combination of ion chromatography (IC) with accelerator mass spectrometry (AMS) was developed to determine the speciation of 14C-(radiocarbon) bearing organic compounds in the femto to pico molar concentration range. The development of this compound-specific radiocarbon analysis (CSRA) of carboxylic acids is reported and the application of the method on a leaching solution from neutron-irradiated steel is demonstrated. The background and the dynamic range of the AMS-based method were quantified. On using 14C-labelled standards, the measurements demonstrate the repeatability of the analytical method and the reproducible recovery of the main target carboxylic acids (i.e., acetate, formate, malonate, and oxalate). The detection limit was determined to be in the mid fmol 14C per L level while the dynamic range of the analytical method covers three orders of magnitude from the low fmol to the mid pmol 14C per L level. Cross contamination was found to be negligible during IC fractionation and was accounted for during eluate processing and 14C detection by AMS. The 14C-bearing carboxylates released from an irradiated steel nut into an alkaline leaching solution were analysed using the CSRA-based analytical method with the aim to check the applicability of the approach and develop appropriate sample preparation. The concentrations of 14C-bearing formate and acetate, the main organic corrosion products, were at a low pmol 14C per L level for convenient dimensions of the alkaline leaching experiment which demonstrates that compound-specific 14C AMS is an extremely sensitive analytical method for analysing 14C-bearing compounds. The content of total organic 14C in solution (TO14C) determined by the direct measurement of an aliquot of the leaching solution agrees well with the sum of the 14C concentrations of the individual carboxylates within the uncertainty of the data. Furthermore, the TO14C content is in good agreement with the calculated value using the corrosion rate determined from the 60Co release and the 14C inventory of the irradiated steel specimen.

Patient-level analysis of incident vancomycin-resistant enterococci colonization and antibiotic days of therapy.

Vancomycin-resistant enterococci (VRE) infections are a public health threat associated with increased patient mortality and healthcare costs. Antibiotic usage, particularly cephalosporins, has been associated with VRE colonization and VRE bloodstream infections (VRE BSI). We examined the relationship between antimicrobial usage and incident VRE colonization at the individual patient level. Prospective, weekly surveillance was undertaken for incident VRE colonization defined by negative admission but positive surveillance swab in a medical intensive care unit over a 17-month period. Antimicrobial exposure was quantified as days of therapy (DOT)/1000 patient-days. Multiple logistic regression was used to analyse incident VRE colonization and antibiotic DOT, controlling for demographic and clinical covariates. Ninety-six percent (1398/1454) of admissions were swabbed within 24 h of intensive care unit (ICU) arrival and of the 380 patients in the ICU long enough for weekly surveillance, 83 (22%) developed incident VRE colonization. Incident colonization was associated in bivariate analysis with male gender, more previous hospital admissions, longer previous hospital stay, and use of cefepime/ceftazidime, fluconazole, azithromycin, and metronidazole (P < 0·05). After controlling for demographic and clinical covariates, metronidazole was the only antibiotic independently associated with incident VRE colonization (odds ratio 2·0, 95% confidence interval 1·2-3·3, P < 0·009). Our findings suggest that risk of incident VRE colonization differs between individual antibiotic agents and support the possibility that antimicrobial stewardship may impact VRE colonization and infection.

The influence of the tricyclic antidepressant amitriptyline on periodic limb movements during sleep.

INTRODUCTION: Many antidepressants are associated with periodic limb movements (PLM) during sleep. Although some tricyclic antidepressants, such as amitriptyline, promote sleep and are thus often prescribed as a treatment for sleep disturbances that can accompany depression, it remains unclear whether amitriptyline is associated with PLM. METHODS: 32 healthy males (18-39 years) spent 2 consecutive nights in the sleep lab for polysomnographic recording. During the second night, they received either 75 mg amitriptyline or placebo in a randomized, double-blind, placebo-controlled manner. RESULTS: In subjects receiving amitriptyline but not in subjects receiving placebo, the number of periodic leg movements per h was significantly increased from baseline to intervention night. However, objective polysomnographic sleep parameters (such as the number of awakenings, wake after sleep onset, and sleep efficiency) and subjective sleep perception were not significantly associated with any PLM indices. DISCUSSION: Our findings indicate that amitriptyline can induce or even increase the number of PLM during sleep in healthy subjects. When treating sleep disturbances with amitriptyline, PLM should be considered as a possible cause of insufficient improvement.

Pneumocystis pneumonia in hospitalized patients: a detailed examination of symptoms, management, and outcomes in human immunodeficiency virus (HIV)-infected and HIV-uninfected persons.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) is a life-threatening infection for immunocompromised individuals. Robust data and clear guidelines are available for prophylaxis and treatment of human immunodeficiency virus (HIV)-related PCP (HIV-PCP), yet few data and no guidelines are available for non-HIV-related PCP (NH-PCP). We postulated that prevention and inpatient management of HIV-PCP differed from NH-PCP. METHODS: We performed a retrospective case review of all pathologically confirmed cases of PCP seen at the University of Alabama Medical Center from 1996 to 2008. Data on clinical presentation, hospital course, and outcome were collected using a standardized data collection instrument. Bivariate analysis compared prophylaxis, adjunctive corticosteroids, and clinical outcomes between patients with HIV-PCP and NH-PCP. RESULTS: Our analysis of the cohort included 97 cases of PCP; 65 HIV and 32 non-HIV cases. Non-HIV cases rarely received primary prophylaxis (4% vs. 38%, P = 0.01) and received appropriate antibiotics later in the course of hospitalization (5.2 days vs. 1.1 days, P < 0.005). Among transplant patients, NH-PCP was diagnosed a mean of 1066 days after transplantation and most patients were on low-dose corticosteroids (87%) at the time of disease onset. No significant differences in adjunctive corticosteroid use (69% vs. 77%, P = 0.39) and 90-day mortality (41% vs. 28%, P = 0.20) were detected. CONCLUSIONS: Patients who have undergone organ or stem cell transplant remain at risk for PCP for many years after transplantation. In our cohort, patients who developed NH-PCP were rarely given prophylaxis, and initiation of appropriate antibiotics was significantly delayed compared to cases of HIV-PCP. Medical providers should be aware of the ongoing risk for NH-PCP, even late after transplantation, and consider more aggressive approaches to both prophylaxis and earlier empirical therapy for PCP.

Observational study of the epidemiology and outcomes of vancomycin-resistant Enterococcus bacteraemia treated with newer antimicrobial agents.

Vancomycin-resistant Enterococcus bloodstream infections (VRE-BSI) are a growing problem with few clinical trials to guide therapy. We conducted a retrospective study of management and predictors of mortality for VRE-BSI at a tertiary-care centre from January 2005 to August 2008. Univariate and multivariable analyses examined the relationship of patient characteristics and antibiotic therapy with 30-day all-cause mortality. Rates of VRE-BSI increased from 0·06 to 0·17 infections/1000 patient-days (P=0·03). For 235 patients, 30-day mortality was 34·9%. Patients were primarily treated with linezolid (44·2%) or daptomycin (36·5%). Factors associated with mortality were haemodialysis [odds ratio (OR) 3·2, 95% confidence interval (CI) 1·6-6·3, P=0·007], mechanical ventilation (OR 3·7, 95% CI 1·3-10·4, P=0·01), and malnutrition (OR 2·0, 95% CI 1·0-4·0, P=0·046). Use of linezolid, but not daptomycin (P=0·052) showed a trend towards an association with survival. In conclusion, VRE-BSI is a growing problem, associated with significant 30-day mortality. Multiple factors were associated with poor outcomes at our hospital.

On species delimitation, hybridization and population structure of cassava whitefly in Africa.

The Bemisia cassava whitefly complex includes species that cause severe crop damage through vectoring cassava viruses in eastern Africa. Currently, this whitefly complex is divided into species and subgroups (SG) based on very limited molecular markers that do not allow clear definition of species and population structure. Based on 14,358 genome-wide SNPs from 62 Bemisia cassava whitefly individuals belonging to sub-Saharan African species (SSA1, SSA2 and SSA4), and using a well-curated mtCOI gene database, we show clear incongruities in previous taxonomic approaches underpinned by effects from pseudogenes. We show that the SSA4 species is nested within SSA2, and that populations of the SSA1 species comprise well-defined south-eastern (Madagascar, Tanzania) and north-western (Nigeria, Democratic Republic of Congo, Burundi) putative sub-species. Signatures of allopatric incipient speciation, and the presence of a 'hybrid zone' separating the two putative sub-species were also detected. These findings provide insights into the evolution and molecular ecology of a highly cryptic hemipteran insect complex in African, and allow the systematic use of genomic data to be incorporated in the development of management strategies for this cassava pest.

Functional imaging of mitochondria in saponin-permeabilized mice muscle fibers.

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTrackerTM Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD-redox system. Under the present experimental conditions these subsets show similar functional responses.

Diet selenium improves the antioxidant defense system of juveniles Nile tilapia ( Oreochromis niloticus L.).

From the advancement of tilapia production in recent years, diets are sought that allow the maximum growth, improving health and fish quality. In this study growth, biochemical, hematological and oxidative parameters were evaluated of tilapia fed with increasing selenium levels: 0.53, 0.86, 1.04 and 1.22 mg kg-1. It was used 400 juveniles (initial weight = 36.51 ± 10.88 g), fed for six weeks. There was no effect of selenium on fish growth, biochemical and hematological parameters. In the oxidative parameters, there was an increase in non-protein thiols and a decrease in malondialdehyde levels, evidencing antioxidant effects of selenium. The diet selenium levels above 0.86 mg kg-1 improved the antioxidant system and does not affect to biochemical, hematological and growth parameters of tilapia juveniles.

Impact of long-term daylight deprivation on retinal light sensitivity, circadian rhythms and sleep during the Antarctic winter.

Long-term daylight deprivation such as during the Antarctic winter has been shown to lead to delayed sleep timing and sleep fragmentation. We aimed at testing whether retinal sensitivity, sleep and circadian rest-activity will change during long-term daylight deprivation on two Antarctic bases (Concordia and Halley VI) in a total of 25 healthy crew members (mean age: 34 ± 11y; 7f). The pupil responses to different light stimuli were used to assess retinal sensitivity changes. Rest-activity cycles were continuously monitored by activity watches. Overall, our data showed increased pupil responses under scotopic (mainly rod-dependent), photopic (mainly L-/M-cone dependent) as well as bright-blue light (mainly melanopsin-dependent) conditions during the time without direct sunlight. Circadian rhythm analysis revealed a significant decay of intra-daily stability, indicating more fragmented rest-activity rhythms during the dark period. Sleep and wake times (as assessed from rest-activity recordings) were significantly delayed after the first month without sunlight (p < 0.05). Our results suggest that during long-term daylight deprivation, retinal sensitivity to blue light increases, whereas circadian rhythm stability decreases and sleep-wake timing is delayed.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1beta- and cAMP-induced nitric oxide synthase expression in rat renal mesangial cells.

Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) regulate mesangial cell proliferation and matrix production in vitro and in vivo and crucially participate in the pathogenesis of glomerulonephritis. We investigated whether PDGF-BB and bFGF influence nitric oxide (NO) production, another important effector molecule in inflammatory glomerular injury. Inducible NO synthase (iNOS) induction in rat glomerular mesangial cells has been described in response to two principal classes of activating signals comprising inflammatory cytokines such as interleukin 1beta (IL-1beta) or elevation of cyclic AMP (cAMP). Treatment of mesangial cells with IL-1beta induces iNOS activity measured as nitrite levels in cell culture supernatants. Coincubation of mesangial cells with PDGF-BB inhibits production of nitrite by approximately 95%. This effect can be reversed by the simultaneous incubation of PDGF-BB in the presence of calphostin C, a potent and selective inhibitor of protein kinase C. In contrast, incubation of cells in the presence of bFGF potentiates IL-1beta-induced production of NO and is functionally associated with an increased rate of apoptosis of mesangial cells. Western blot analyses reveal that PDGF-BB causes a decrease in the formation of iNOS protein which is preceded by decreases in iNOS mRNA steady state levels. bFGF drastically increases iNOS protein levels as well as the corresponding iNOS mRNA steady state levels. Nuclear run-on experiments reveal that PDGF-BB decreases the IL-1beta-induced transcription rate of the iNOS gene, whereas bFGF potentiates the transcriptional activity of the iNOS gene. Northern blot analyses demonstrate that bFGF strongly potentiates the formation of IL-1beta-induced IL-1 type I receptor mRNA levels, whereas PDGF-BB has no effect. Treatment of mesangial cells with the membrane-permeable cAMP analogue N6, O-2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP) markedly increases the production of nitrite. Whereas PDGF-BB does not affect cAMP-induced nitrite levels, bFGF strongly potentiates them. PDGF-BB alters neither cAMP-induced iNOS protein levels nor the corresponding iNOS mRNA steady state levels. By contrast, bFGF superinduces cAMP-stimulated iNOS protein and iNOS mRNA levels. These changes by bFGF are due to an increase in cAMP-induced transcriptional activity of the iNOS gene which is not affected by PDGF-BB. In summary, the results show that PDGF and bFGF differentially regulate iNOS expression in mesangial cells in a stimulus-specific way. The timely sequence of expression of PDGF and bFGF and of cytokines like IL-1 will crucially determine the amounts of NO produced and the functional consequences thereof in the course of progressive glomerular diseases.

Self-reported empathic abilities in schizophrenia.

BACKGROUND: Increasing evidence indicates impairments of empathic abilities in schizophrenia that may impact outcome and course of the disease. While there is consensus on the presence of deficits in 'theory of mind' in this disorder, i.e. cognitive aspects of mental state attribution, the ability to infer emotional experiences of others, i.e. affective empathy, has not been investigated so far. METHODS: We assessed multiple dimensions of empathy in 45 schizophrenic patients and 45 healthy controls, matched for age and gender, with a self-rating instrument, the Interpersonal Reactivity Index (IRI). To control for modulating effects of cognitive deficits, a neuropsychological test battery was employed. RESULTS: Schizophrenic patients showed significantly lower scores in cognitive empathy ('perspective taking': F=12.176, df=1, p=0.001) but more self-related aversive feelings in response to the distress of others ('personal distress: F=16.477, df=1, p<0.001). Self-ratings of affective empathy, i.e. concern for others, did not differ between groups. Results in the domains of empathy were not explained by symptoms or neurocognition as revealed by regression analysis. However, lower scores in 'perspective taking' were found with advancing duration of illness (r=-0.453, p=0.002). CONCLUSIONS: Results indicate reductions of cognitive empathy but relatively preserved emotional empathic abilities in schizophrenia. Although previous studies observed deficits in emotion perception and expression, our findings support the concept of differentially disturbed abilities in cognitive and emotional empathy in schizophrenia.

Can short-wavelength depleted bright light during single simulated night shifts prevent circadian phase shifts?

In single night shifts, extending habitual wake episodes leads to sleep deprivation induced decrements of performance during the shift and re-adaptation effects the next day. We investigated whether short-wavelength depleted (=filtered) bright light (FBL) during a simulated night shift would counteract such effects. Twenty-four participants underwent a simulated night shift in dim light (DL) and in FBL. Reaction times, subjective sleepiness and salivary melatonin concentrations were assessed during both nights. Daytime sleep was recorded after both simulated night shifts. During FBL, we found no melatonin suppression compared to DL, but slightly faster reaction times in the second half of the night. Daytime sleep was not statistically different between both lighting conditions (n = 24) and there was no significant phase shift after FBL (n = 11). To conclude, our results showed positive effects from FBL during simulated single night shifts which need to be further tested with larger groups, in more applied studies and compared to standard lighting.

Hippocampal glutamate concentration predicts cerebral theta oscillations during cognitive processing.

RATIONALE: Brain waves reflect collective behavior of neurons and provide insight into distributed network processing. Frontal and hippocampal theta oscillations (4-7 Hz) were linked to cognitive tasks and animal studies have suggested an involvement of glutamatergic neurotransmission in integrative frontal-hippocampal processing. Human evidence for such relationships is lacking. METHODS: Here, we studied the associations between glutamate concentrations in the hippocampal region, measured by a 3-T proton magnetic resonance spectroscopy (1H-MRS), and EEG theta activity during an auditory target detection paradigm. RESULTS: A robust relationship between hippocampal glutamate and frontal theta activity during stimulus processing was found. Moreover, frontal theta oscillations were related to response speed. CONCLUSION: The results suggest a functional coupling between the frontal cortex and hippocampal region during stimulus processing and support the idea of the hippocampus as a neural rhythm generator driven by glutamatergic neurotransmission. These preliminary data show, for the first time, a relationship between in vivo measured glutamate and basic cerebral information processing in humans.

EXAFS study of U(VI) uptake by calcium silicate hydrates.

Among the different cement minerals, calcium silicate hydrates (C-S-H) are the prime candidates for heavy metal binding because of their abundance and appropriate structure. Immobilization processes of heavy metals by cementitious materials, and in particular C-S-H phases, thus play an important role in multibarrier concepts developed worldwide for the safe disposal of hazardous and radioactive wastes. In this study, the uptake of U(VI) by C-S-H has been investigated using X-ray absorption fine structure (XAFS) spectroscopy. C-S-H phases were synthesized using two different procedures: One is based on the mixing of CaO and SiO2 solids ("direct reaction" method); for the other one starting solutions of Ca and Si are used ("solution reaction" method). XAFS investigations were carried out on samples doped with U(VI). U(VI) was either sorbed onto previously precipitated C-S-H phases (sorption samples) or added during C-S-H synthesis (coprecipitation samples). The coordination environment of U(VI) in the sorption samples was found to be independent of the procedure used for C-S-H synthesis. A split equatorial oxygen shell (Oeq1: R=2.23-2.27 A; Oeq2: R=2.36-2.45 A), neighboring silicon atoms at short (R=3.07-3.11 A) and long (R=3.71-3.77 A) distances, and neighboring Ca atoms (R=3.77-3.81 and 4.15-4.29 A) were observed for all the samples. The structural parameters resemble those reported for uranophane. The coordination environment of U(VI) in the coprecipitation samples depends on the method used for C-S-H synthesis, and further, the spectra differ from those determined for the sorption samples. UU backscattering contributions were observed in the samples prepared using the direct reaction method, whereas no split equatorial shell appeared in the samples prepared using the solution reaction method.

A novel serine-dependent proteolytic activity is responsible for truncated signal transducer and activator of transcription proteins in acute myeloid leukemia blasts.

Hematopoietic cytokine receptor signaling involves activation of signal transducer and activator of transcription (STAT) proteins that are thought to control cellular differentiation. Truncated STAT isoforms (beta forms, rather than the normal alpha forms) have been described and found to block the normal signaling function of the alpha isoforms. We recently demonstrated STATbeta isoforms in bone marrow samples from 21 of 27 (78%) acute myeloid leukemia (AML) patients. We sought to determine the mechanism by which the STATbeta forms were generated. Samples from eight newly diagnosed AML patients were studied; four expressed predominantly STATalpha, and four expressed predominantly STATbeta. The reverse transcription-PCR generated identical products in the two groups, suggesting that alternate mRNA splicing is not responsible for the genesis of STATbeta. Extracts from cells expressing predominantly STATbeta incubated with cell extracts from the MO7E cell line, which expresses predominantly STATa, caused a decrease of the alpha isoforms and an increase of the beta isoforms, suggesting the presence of proteolytic activity. This proteolytic activity was: (a) specific for STAT3 and STAT5, but not for STAT6; (b) serine dependent; (c) equally present in nuclear and cytoplasmic fractions of the leukemic blasts; and (d) different than the activity detected in a murine hematopoietic cell line. The cleaved beta isoforms retained their DNA-binding activity. Because expression of truncated STATs may be involved in blocking differentiation of AML blasts, elucidation of the regulation of the proteolytic activity may contribute to our understanding of leukemogenesis.

Energy metabolism in rat pancreatic acinar cells during anoxia and reoxygenation.

Mitochondrial energy metabolism was studied in isolated pancreatic acinar cells during anoxia up to 90 min and reoxygenation for 60 min. To identify critical alterations leading to the known postanoxic impairments in structure and function of acinar cells, adenine nucleotide levels and the rates of phosphorylating and non-phosphorylating respiration were determined. ATP levels and the total amount of adenine nucleotides strongly decreased as early as after 30 min of anoxia. The cells partially restored ATP and the total adenine nucleotides within 60 min of reoxygenation. Long-term anoxia caused an increase in the oligomycin-insensitive part of oxygen consumption. The respiratory capacity measured as uncoupled respiration progressively declined to 40% of controls after 90 min of anoxia. Fluorescence measurements showed that flavoproteins and mitochondrial pyridine nucleotides in reoxygenated cells after short-term anoxia were in a more reduced state than in aerobic controls, and were not fully oxidizable by uncoupling. It is concluded that long-term anoxia produces an irreversible loss of respiratory capacity leading to a limited ATP production. This functional impairment and the progressive damage to acinar cells may be relevant for pancreatic injuries such as acute pancreatitis or post-transplantation pancreatitis.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1beta- and cAMP-induced group II phospholipase A2 expression in rat renal mesangial cells.

Expression of group II phospholipase A2 (PLA2; EC 3.1.1.4) in rat renal mesangial cells is triggered in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin 1beta (IL-1beta) or tumor necrosis factor alpha and agents that elevate cellular levels of cyclic AMP (cAMP) such as forskolin, an activator of adenylate cyclase. Treatment of mesangial cells with IL-1beta or forskolin for 24 h induces group II PLA2 activity secreted into cell culture supernatants by about 15-fold and 11-fold, respectively. Platelet-derived growth factor (PDGF)-BB potently inhibits secretion of IL-1beta- and forskolin-induced group II PLA2 activity. By Western and Northern blot analyses, we demonstrate that this is due to a reduction of PLA2 protein levels and the corresponding PLA2 mRNA steady-state levels. Basic fibroblast growth factor (bFGF) virtually does not inhibit IL-1beta-stimulated group II PLA2 activity, but markedly inhibits forskolin-induced expression of group II PLA2 activity. These effects are caused by changes in the corresponding PLA2 protein and PLA2 mRNA steady-state levels. Inhibition of protein kinase C (PKC) by the potent and selective PKC inhibitor calphostin C converted the inhibitory action of PDGF into a bFGF-type of response thus suggesting that PKC is a major effector in PDGF-induced inhibition of IL-1beta-stimulated group II sPLA2 expression. In summary, our data suggest that PDGF and bFGF differentially modulate in a stimulus-specific manner the expression of group II PLA2 in mesangial cells.

Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I.

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-gamma-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501-532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.

Evaluation of serum levels of solubilized adhesion molecules and cytokine receptors in coronary heart disease.

OBJECTIVES: The diagnostic importance of circulating solubilized tumor necrosis factor-alpha receptor II (sTNF-alphaRII) and interleukin-2 receptor in coronary heart disease (CHD) was evaluated. In addition, these variables were correlated with solubilized adhesion molecule levels (i.e., intracellular adhesion molecule [ICAM], vascular cell adhesion molecule [VCAM], and E-selectin). BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory process. Because the immunologic network presents an abundance of positive and negative feedback mechanisms, information obtained from different immunologic variables might be highly redundant. METHODS: In a cross-sectional study design, 60 patients with angiographically proven CHD were compared with 60 individuals who had undergone coronary angiography but in whom no evidence of stenosis could be found (control subjects). Angiography was performed at least one year before the study. Cytokine levels were determined by enzyme-linked immunosorbent assay technique and evaluated by univariate and multivariate statistical methods. RESULTS: All immunologic variables except E-selectin (p = 0.08) significantly discriminated between patients and control subjects. Coronary artery bypass graft surgery after angiography did not lead to significant differences in the variables investigated in the patients with bypass surgery as compared with the subjects without bypass surgery. Receiver-operating characteristics analysis showed comparable test accuracy for solubilized adhesion molecules and cytokine receptors. Multivariate logistic regression analysis, including age, revealed that only ICAM and sTNF-alphaRII were independently correlated with the presence of CHD. Patients belonging to the third tertile of at least one of these two variables demonstrated a 1.6- to 2-fold increased relative risk for the presence of CHD. CONCLUSIONS: We concluded that both circulating ICAM and TNF-alphaRII should be further evaluated as markers for atherosclerotic changes in the coronary system.