Decreased exhaled nitric oxide as a marker of postinsult immune paralysis.

Nitric oxide (NO) regulates neutrophil migration and alveolar macrophage functions such as cytokine synthesis and bacterial killing, both of which are impaired in immune paralysis associated with critical illness. The aim of this study was to determine whether NO is involved in immune paralysis and whether exhaled NO measurement could help to monitor pulmonary defenses. NO production (protein expression, enzyme activity, end products, and exhaled NO measurements) was assessed in rats after cecal ligation and puncture to induce a mild peritonitis (leading to approximately 20% mortality rate). An early and sustained decrease in exhaled NO was found after peritonitis (from 1 to 72 h) compared with healthy rats [median (25th-75th percentile), 1.5 parts per billion (ppb) (1.2-1.7) vs. 4.0 ppb (3.6-4.3), P < 0.05], despite increased NO synthase-2 and unchanged NO synthase-3 protein expression in lung tissue. NO synthase-2 activity was decreased in lung tissue. Nitrites and nitrates in supernatants of isolated alveolar macrophages decreased after peritonitis compared with healthy rats, and an inhibitory experiment suggested arginase overactivity in alveolar macrophages bypassing the NO substrate. Administration of the NO synthase-2 inhibitor aminoguanidine to healthy animals reproduced the decreased neutrophil migration toward alveolar spaces that was observed after peritonitis, but L-arginine administration after peritonitis failed to correct the defect of neutrophil emigration despite increasing exhaled NO compared with D-arginine administration [4.8 (3.9-5.7) vs. 1.6 (1.3-1.7) ppb, respectively, P < 0.05]. In conclusion, the decrease in exhaled NO observed after mild peritonitis could serve as a marker for lung immunodepression.     

SOME PRYMNESIACEAE (HAPTOPHYTA,PRYMNESIOPHYCEAE) FROM THE MEDITERRANEAN SEA,WITH THE DESCRIPTION OF TWO NEW SPECIES: CHRYSOCHROMULINA LANCEOLATA SP. NOV. AND C. PSEUDOLANCEOLATA SP. NOV.1

Five species belonging to the family Prymnesiaceae (one Prymnesium and four Chrysochromulina) have been identified in cultures obtained from water collected in the Bay of Banyuls‐sur‐Mer (Mediterranean Sea, France) using LM, SEM, and TEM. Two are described as new species, Chrysochromulina lanceolata sp. nov. and C. pseudolanceolata sp. nov. Both species are large and lanceolate with an acute posterior and two anterior arms. They are easily detectable with LM but difficult to distinguish to species level with live cells, without experience. EM reveals two completely different scale patterns in the two species. Cells of C. lanceolata are 21–38 μm long, 7–12 μm wide, and 3–7 μm thick. They possess two subequal flagella (30–51 and 29–44 μm), and the haptonema is shorter than the flagella (23–37 μm). The cell body is covered by plate and spine scales. Cells of C. pseudolanceolata sp. nov. are slightly smaller (15–18 × 6–8 μm) with more rounded extremities, two subequal flagella (19–26 and 17–24 μm), and the haptonema is longer than the flagella (about 35 μm). Three types of plate scales are observed in this species. Other findings are C. alifera Parke et Manton and C. throndsenii Eikrem (a new record for the Mediterranean Sea). Prymnesium faveolatum Fresnel, a new toxic species recently described, is illustrated with both LM and SEM.     

S-decyl-glutathione nonspecifically stimulates the ATPase activity of the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 (ABCC1).

The human multidrug resistance-associated protein(MRP1) is an ATP-dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well-characterized multidrug transporter P-gp, MRP1 comprises two nucleotide-binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P-gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione-S-transferase (GST) or the maltose-binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST-fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent K(m) of 340 microm and a V(max) of 6.0 nmol P(I) x mg-1 x min-1, and a K(m) of 910 microm ATP and a V(max) of 7.5 nmol P(I) x mg-1 x min-1, respectively. Remarkably, S-decyl-glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S-decyl-glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations.     

Passive properties of the diaphragm in COPD.

Structural adaptations that occur in the diaphragm muscle of patients with chronic obstructive pulmonary disease (COPD), namely an increase in type I fibers and a decrease in type II fibers, have been explored in terms of the active contractile properties of the diaphragm. The aim of this study was to test the passive properties of the diaphragm by measuring the force response of relaxed diaphragm muscle fibers to stretching to determine the effect of COPD on these properties. Costal diaphragm biopsies were taken from patients with COPD and from controls with normal pulmonary function. From these biopsies, titin expression was assessed in diaphragm homogenates by gel electrophoresis, and the restoring force was measured by incremental stretching of single fibers in the relaxed state and measuring the force response to stretching. A quadratic model was used to illustrate the relationship between restoring force and muscle fiber length, and it revealed that COPD fibers generate significantly lower restoring forces than control fibers as judged by the area under the force-length curve. Furthermore, this finding applies to both type I and type II fibers. Gel electrophoresis revealed different titin isoforms in COPD and controls, consistent with the conclusion that COPD results not only in a change in muscle fiber-type distribution but in a structural change in the titin molecule in all muscle fiber types within the diaphragm. This may assist the muscle with the energetic changes in the length of the diaphragm required during breathing in COPD.     

Activity Pattern of the Trawling Phyllostomid Bat,Macrophyllum macrophyllum,in Panamá1

We studied activity patterns of long‐legged bats, Macrophyllum macrophyllum (Phyllostomidae), in the Barro Colorado Nature Monument, Panamá, using radio‐telemetry. Activity of four males and five females equipped with radio‐transmitters were monitored for 4–7 entire nights each between April and July 2002. Bats exhibited maximum activity around dusk and high activity during the night. Males and females foraged for equal amounts of time in continuous flight (mean: 7 min, maximum 1 h) with interspersed resting phases (mean: 15 min, maximum 3 h). Activity of M. macrophyllum was sensitive to several factors. Time of emergence and return to day roost were correlated with time of sunset and sunrise, respectively. Maximum bat activity coincided with high abundance of aerial insects. Finally, heavy rain caused bats to reduce or cease flight activity. Direct observations and field video recordings support the assumption that M. macrophyllum employs two distinct foraging modes: trawling of insects from and capture of aerial insects at low heights above water. Combination of foraging modes gives M. macrophyllum high flexibility and efficiency in prey search. Activity, foraging mode, and morphology, which are similar to trawling bats from other families, distinguish M. macrophyllum from all other phyllostomid species and grant it access to open habitat above water, a habitat no other phyllostomid bat has conquered.     

Curcumin induced suicidal erythrocyte death.

The natural nutrient component Curcumin with anti-inflammatory and antitumor activity has previously been shown to stimulate apoptosis of several nucleated cell types. The present study has been performed to explore whether Curcumin could similarly induce suicidal death of erythrocytes or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing cells are phagocytosed and thus rapidly cleared from circulating blood. Erythrocyte membrane scrambling may be triggered by increase of cytosolic Ca(2+) activity or formation of ceramide. To test for eryptosis, erythrocyte phosphatidylserine exposure has been estimated from annexin V binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to Curcumin (= 1 microM) increased annexin V binding and decreased forward scatter, pointing to phosphatidylserine exposure at the cell surface and cell shrinkage. According to Fluo3 fluorescence Curcumin increased cytosolic Ca(2+) activity and according to immunofluorescence Curcumin increased ceramide formation. As shown previously, hypertonic shock (addition of 550mM sucrose), chloride removal and glucose depletion decreased the forward scatter and increased annexin V binding. The effects on annexin binding were enhanced in the presence of Curcumin. Exposure to Curcumin did, however, not significantly enhance the shrinking effect of hypertonic shock or Cl(-) removal and reversed the shrinking effect of glucose withdrawal. The present observations disclose a proeryptotic effect of Curcumin which may affect the life span of circulating erythrocytes.     

Effect of Bafilomycin A1 and Nocodazole on Endocytic Transport in HeLa Cells: Implications for Viral Uncoating and Infection

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used ³⁵S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713–3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.