Thermodynamic phase plane analysis of ventricular contraction and relaxation

Background  

Ventricular function has conventionally been characterized using indexes of systolic (contractile) or diastolic (relaxation/stiffness) function. Systolic indexes include maximum elastance or equivalently the end-systolic pressure volume relation and left ventricular ejection fraction. Diastolic indexes include the time constant of isovolumic relaxation - and the end-diastolic pressure-volume relation. Conceptualization of ventricular contraction/relaxation coupling presents a challenge when mechanical events of the cardiac cycle are depicted in conventional pressure, P, or volume, V, terms. Additional conceptual difficulty arises when ventricular/vascular coupling is considered using P, V variables.     

Tumour necrosis factor alpha, lipid peroxidation and NO* are increased and associated with decreased free-radical scavenging enzymes in patients with Weill-Marchesani syndrome

AIM: Weill-Marchesani syndrome (WMS) is a rare systemic disorder with both autosomal recessive and dominant inheritances. Accumulation of reactive oxygen species such as O2*-, H2O2 and OH* causes lipid peroxidation (LPO), whereas antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GSHPx)) mediate defence against oxidative stress. Excess tumour necrosis factor (TNF)-alpha and NO* react with O2*- and cause further antioxidant depletion with an increase in mutation frequency by H2O2. This study investigated the levels of SOD, GSHPx, catalase (CAT), TNF-alpha, NO and LPO in patients with WMS. METHODS: A group of 10 WMS patients (four males, six females; age, 26.5+/-19.0 years) and 10 age-matched and sex-matched controls (five males, five females; age, 27.3+/-18.2 years) were included. Serum TNF-alpha levels were determined by a spectrophotometer technique using immulite chemiluminescent immunometric assay. Malondialdehyde (MDA) was determined in plasma; CAT in red blood cells (RBCs), and SOD and GSHPx in both plasma and RBCs. Total serum NO* levels were evaluated by Griess reaction. RESULTS: Mean levels of TNF-alpha (8.3+/-0.6 pg/ml) in WMS patients were significantly (p<0.001) higher than controls (4.3+/-0.2 pg/ml). Plasma MDA levels in patients and controls were 5.4+/-0.8 and 1.8+/-0.6 micromol/l, respectively, and the difference was significant (p=0.0002). SOD and GSHPx activities were significantly lower in both RBCs and plasma of WMS than in controls (RBC-SOD, 3981.9+/-626.6 versus 5261.6+/-523.0 U/g haemoglobin (Hb), p=0.0005; plasma-SOD, 529.4+/-49.3 versus 713.4+/-55.7 U/g protein, p=0.0002; RBC-GSHPx, 682.7+/-42.0 versus 756.5+/-47.6 U/g Hb, p=0.0011; plasma-GSHPx, 107.3+/-15.0 versus 131.4+/-19.7 U/g protein, p=0.0113). In addition, serum NO (NO*-2 + NO*-3) levels were also significantly (p = 0.0002) increased in WMS patients (54.4+/-5.7 versus 26.9+/-6.7 micromol/l). RBC-CAT levels were similar between groups (125.6+/-21.3 versus 131.0+/-21.5 k/g Hb, p = 0.8798). CONCLUSIONS: The elevated LPO, TNF-alpha and NO* with decreased antioxidant enzyme activities indicated impaired antioxidative defence mechanisms with an oxidative injury and cell toxicity in WMS patients. The use of multiple antioxidants and free radical scavengers might be helpful in this genetic disorder.     

Postnatal glucocorticoids induce alpha-ENaC formation and regulate glucocorticoid receptors in the preterm rabbit lung

At birth, lung fluid clearance is coupled to Na+ transport through epithelial Na+ channels (ENaC) in the distal lung epithelium. We evaluated the effect of postnatal glucocorticoids (GC) on lung alpha-ENaC expression in preterm 29-day gestational age (GA) fetal rabbits. Postnatal treatment of 29-day GA fetuses with 0.5 mg/kg of dexamethasone (Dex) iv resulted in a 2- and 22-fold increase in lung alpha-ENaC mRNA expression compared with saline-treated fetuses after 8 and 16 h, respectively. Lung alpha-ENaC protein levels in Dex-treated fetuses were also elevated compared with saline-treated counterparts. The extravascular lung water (EVLW)/dry lung tissue weight ratios of 29-day GA fetuses treated with either saline or Dex decreased over 24 h compared with that observed at birth; however, at 24 h, the EVLW/dry lung tissue weight ratios of saline- and Dex-treated fetuses were similar. Dex-induced alpha-ENaC mRNA and protein levels were attenuated by glucocorticoid receptor (GCR) antagonist RU-486 in fetal distal lung epithelial cells isolated from 29-day GA fetuses, indicating that GC-dependent augmentation of lung alpha-ENaC requires the presence of functional GCR. Lung GCR mRNA expression and protein levels were elevated in 29-day GA fetuses compared with fetuses at earlier GA. Exposure of 29-day GA fetuses to Dex for 16 h caused a 2.1-fold increase in lung GCR mRNA expression, but GCR protein levels were decreased in Dex-treated fetuses after 24 h. We conclude that postnatal treatment of preterm 29-day GA fetal rabbits with GC results in an elevation of lung alpha-ENaC accompanied by an autoregulation of pulmonary GCR.     

Comparison of two different stool antigen tests for the primary diagnosis of Helicobacter pylori infection in turkish patients with dyspepsia

AIM: To assess the reliability of two different enzyme immunoassays in detecting the Helicobacter pylori status in stool specimens of Turkish patients with dyspepsia. MATERIALS AND METHODS: One hundred and fifty-one patients [74 with nonulcer dyspepsia (NUD), 64 with duodenal ulcer (DU) and 13 with gastric cancer] who were admitted to the endoscopy unit of Istanbul University, Cerrahpasa Medical Faculty for upper gastrointestinal endoscopy because of dyspepsia were enrolled in the study. Helicobacter pylori infection was confirmed in all patients by histology, rapid urease test and culture. A patient was classified as being H. pylori-positive if the culture alone or both the histology and the rapid urease test were positive and as negative only if all of these tests remained negative. Stool samples were obtained from patients to assess the reliability of a monoclonal (FemtoLab H. pylori) and a polyclonal (Premier Platinum HpSA) stool antigen test and to compare the diagnostic accuracies of these two tests. A chi2 test was used for statistical comparisons. RESULTS: Using cut-off values of 0.19 for FemtoLab H. pylori and 0.16 for Premier Platinum HpSA, the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were 93%, 90%, 98%, 68% and 93% for the monoclonal test and 84%, 67%, 94%, 40% and 81% for the polyclonal test, respectively. The sensitivity, specificity, negative predictive value and diagnostic accuracy of the monoclonal test were significantly greater than those of the polyclonal test (chi2 = 3.98; p < .05 for sensitivity and chi2 = 15.67; p = .000 for specificity, chi2 = 15.78; p = .000 for negative predictive value and chi2 = 6.37; p = .012 for diagnostic accuracy). The bacterial load did not affect the sensitivity of either test. CONCLUSIONS: The monoclonal FemtoLab H pylori test, using a cut-off 0.19, is a very sensitive, specific and easy to perform diagnostic tool for the primary diagnosis of H. pylori infection in Turkish patients with dyspepsia.     

Arabidopsis thaliana Ogg1 protein excises 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from oxidatively damaged DNA containing multiple lesions

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thalianahas recently been cloned, isolated, and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

Cytotoxic factor-autoantibodies: possible role in the pathogenesis of dengue haemorrhagic fever

During dengue virus infection a unique cytokine, cytotoxic factor (hCF), is produced that is pathogenesis-related and plays a key role in the development of dengue haemorrhagic fever (DHF). However, what regulates the adverse effects of hCF is not known. We have previously shown that anti-hCF antibodies raised in mice, neutralise the pathogenic effects of hCF. In this study we have investigated the presence and levels of hCF-autoantibodies in sera of patients with various severity of dengue illness (n=136) and normal healthy controls (n=50). The highest levels of hCF-autoantibodies (mean+/-S.D.=36+/-20 U ml(-1)) were seen in patients with mild illness, the dengue fever (DF), and 48 out of 50 (96%) of the sera were positive. On the other hand the hCF-autoantibody levels declined sharply with the development of DHF and the levels were lowest in patients with DHF grade IV (mean+/-S.D.=5+/-2 U ml(-1); P=<0.001 as compared to DF). Only one of the 13 DHF grade IV patients had an antibody level above the 'cut-off' value (mean plus 3 S.D. of the control sera). The analysis of data with respect to different days of illness further showed that the highest levels of hCF-autoantibodies were present in DF patients at >9 days of illness. Moreover, the DF patients at all time points, i.e. 1-4, 5-8 and >9 days of illness had significantly higher levels of hCF-autoantibodies (P<0.001) than patients with DHF grade I, II, III and IV. In addition DHF grade I and grade II patients had significantly more positive specimens than DHF grade III and grade IV patients at all time points. These results suggest that elevated levels of hCF-autoantibodies protect the patients against the development of severe forms of DHF and, therefore, it may be useful as a prognostic indicator.     

Impact of cell culture process changes on endogenous retrovirus expression

Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary.     

Flavonoid glycosides from Centaurea pseudoscabiosa subsp. pseudoscabiosa from Turkey

Three flavonoid glycosides were isolated and characterized, together with a further 13 substances belonging to various classes of compounds, in particular two phenolic acids, a coumarin, a sugar and nine flavonoids from the flowered aerial parts of Centaurea pseudoscabiosa subsp. pseudoscabiosa Boiss. et Buhse (Asteraceae). Some considerations about their evolutionary meaning are provided.