Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism

In this study, we show that oxygen regulates nitric oxide (NO) levels through effects on NO synthase (NOS) enzyme kinetics. Initially, NO synthesis in the static lung was measured in bronchiolar gases during an expiratory breath-hold in normal individuals. NO accumulated exponentially to a plateau, indicating balance between NO production and consumption in the lung. Detection of NO2-, NO3-, and S-nitrosothiols in lung epithelial lining fluids confirmed NO consumption by chemical reactions in the lung. Interestingly, alveolar gas NO (estimated from bronchiolar gases at end-expiration) was near zero, suggesting NO in exhaled gases is not derived from circulatory/systemic sources. Dynamic NO levels during tidal breathing in different airway regions (mouth, trachea, bronchus, and bronchiole) were similar. However, in individuals breathing varying levels of inspired oxygen, dynamic NO levels were notably dependent on O2 concentration in the hypoxic range (KmO2 190 microM). Purified NOS type II enzyme activity in vitro was similarly dependent on molecular oxygen levels (KmO2 135 microM), revealing a means by which oxygen concentration affects NO levels in vivo. Based upon these results, we propose that NOS II is a mediator of the vascular response to oxygen in the lung, because its KmO2 allows generation of NO in proportion to the inspired oxygen concentration throughout the physiologic range.     

Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescei n (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83-87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054) and the photochemical cross-linking data.     

Erythropoiesis and erythropoietin synthesis during aseptic acute inflammation

Erythropoietin (Epo) production during acute inflammation induced by s. c. turpentine administration in experimental Long-Evans rats increased in response to reduced erythropoiesis. A close correlation was found between decreased haematocrit (Hct) and increased levels of tumour necrosis factor-alpha (TNF alpha) in this experimental system. The Epo response was not different between rats with acute inflammation and anaemia and control animals with a comparable degree of anaemia. It is concluded that Epo is not an acute phase reactant, and that the Epo response in acute experimental inflammation in rats is explained by the associated development of anaemia.     

The triplet state of the FMO complex of the green sulfur bacterium Prosthecochloris aestuarii studied with single-crystal EPR

Triplet-electron paramagnetic resonance (EPR) spectra were obtained of single crystals of the FMO complex of the green sulfur bacterium Prostecochloris aestuarii. The experiments support the results presented in a previous paper (Louwe et al., J. Phys. Chem. 101 (1997) 11280), which showed that the experimental optical spectra of this pigment-protein complex are best reproduced by assuming that one bacteriochlorophyll (BChl 3) is energetically isolated and that this BChl is the triplet-carrying BChl of the FMO complex at cryogenic temperatures for low excitation density. When comparing the experimental and simulated data sets of the triplet-EPR spectra in single crystals, the best fit is obtained for two triplet states, one localized at BChl 3 and the other at BChl 1. The existence of two different triplet states is traced to the relatively high excitation power necessary to observe the small triplet-EPR signal of the FMO single crystals.     

Arthralgias following dilation and curettage

The paper presents the results of examination of 110 alcoholic patients who have committed criminal actions and were recognized as irresponsible at forensic examination. It was established that wide spectrum of mental disorders were present in such cases--from superacute psychotic states (15 patients) and acute disorders (49) to chronic psychoses (33) and encephalopathy (13). According to clinical manifestations mental disorders correspond in such cases to reactions of exogenic type. In contrast to general medical departments where patients with alcoholic delirium prevail, the studied sample of patients had primarily psychoses with hallucinative-delirious and delirious disorders. Disorders of personality manifested as typical alcoholic, asthenoneurotic, psychopathic-like, residual-psychotic, psychoorganic changes and partial dementia (19 cases).     

Virtual colonoscopy: imaging features with colonoscopic correlation

BACKGROUND: We determined whether activation of the nitric oxide/cyclic guanosine monophosphate pathway by sodium nitroprusside (SNP) protects hearts subjected to cardioplegic arrest and prolonged hypothermic storage. METHODS: Isolated rat hearts arrested with St. Thomas' II cardioplegia and stored at 3 degrees +/- 1 degree C for 8 hours were reperfused at 37 degrees C in Langendorff (10 minutes) and working (60 minutes) modes. RESULTS: During reperfusion, left ventricular work was depressed in stored hearts relative to fresh hearts. When present during arrest, storage, and both reperfusion phases, SNP (200 mumol/L) improved work to values close to those in fresh hearts. When added only during the 10-minute period of Langendorff reperfusion, SNP also improved the subsequent recovery of work. This effect was antagonized by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Poststorage coronary perfusion was not increased by SNP. CONCLUSIONS: The ability of SNP to enhance recovery independent of changes in coronary perfusion and in an ODQ-sensitive manner suggests that SNP-induced protection is due to activation of the myocardial nitric oxide/cyclic guanisine monophosphate pathway. These results suggest that supplementing cardioplegic solutions with SNP, administering SNP during early reperfusion, or both may offer additional means to improve donor heart preservation.     

EPR investigation of compound I in Proteus mirabilis and bovine liver catalases: formation of porphyrin and tyrosyl radical intermediates

Compound I of Proteus mirabilis and bovine liver catalases (PMC and BLC, respectively) were studied combining EPR spectroscopy and the rapid-mix freeze-quench techniques. Both enzymes, when treated with peroxyacetic acid, form a catalytic intermediate which consists of an oxoferryl porphyrin pi-cation radical. In PMC this intermediate is semistable, and an unexpected reversible equilibrium under pH influence takes place between two forms of compound I with different coupling between the oxoferryl and the porphyrin pi-cation radical. At acid pH, one form has a ferromagnetic character as in Micrococcus luteus compound I. At neutral pH, another form with a much smaller coupling, reminiscent of the horse radish peroxidase compound I, is detected. The approximate midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pKa value of an histidyl residue. The residues possibly involved in the transformation are discussed in terms of the known structure of PMC compound I. The EPR spectrum of BLC compound I (pH 5.6), obtained in the millisecond time scale (40 ms), also showed a mixture of two forms which, most probably, correspond to two different magnetic exchange interactions, as in the case of PMC. Taken together, the low-temperature electronic absorption and the EPR spectra of BLC compound I formed in the 0.04-15 s range show that the porphyrin pi-cation radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to the C2,6 and C3,5 ring protons and the beta-methylene protons of the purported tyrosyl radical. Candidates for such a tyrosyl radical are discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor.     

Sustained dapsone-induced remission of hypocomplementemic urticarial vasculitis--a case report

Hypocomplementemic urticarial vasculitis (HUV) is often misdiagnosed. The response to drug therapy may be unsatisfactory, and immunosuppressive therapy may be associated with significant side effects. A 35-year-old patient whose condition was resistant to cyclophosphamide, corticosteroids, pentoxyphylline, cyproheptadine, sulindac, and colchicine was maintained in remission with dapsone, which may be the drug of choice for HUV. Emphysema has been reported to complicate HUV, but this is the first account of vasculitis-related emphysema with no confounding history of tobacco smoke exposure. The relationship of proteolytic injury to the pulmonary and other manifestations is considered, as is the possible role for dapsone in reducing it.