Replicative activity of lung type 2 cells following lung X irradiation

We investigated the replicative activity of type 2 cells in the lungs of mice at various times from 3 to 22 weeks after 18 Gy of X rays to the thorax. No significant changes were found until 11 weeks after thoracic X irradiation. Thereafter the replicative index of type 2 cells was significantly elevated, rising four to sixfold above that of control, sham-irradiated mice. During the period when the replicative activity of type 2 cells was elevated, the breathing frequency increased and there was histologic evidence of the presence of radiation pneumonitis. The magnitude of each of these indices of pneumonitis correlated significantly with the type 2 cell replicative index, suggesting that type 2 cell replication is related to pneumonitis in extent as well as in chronology. How these changes relate to the pathogenesis of radiation pneumonitis is unclear.     

Interaction of fatty acid sodium salts with sodium deoxycholate

Interaction of homologous fatty acids (C3-C18) with sodium deoxycholate was investigated. From NMR and ultrasonic results it was found that short chain homologues (up to C9) do not participate in the formation of mixed micelles with sodium deoxycholate. Fatty acid homologues with longer chains (starting with C9) form mixed micelles by "burying" hydrophobic chains in hydrophobic environment of a sodium deoxycholate micelle.     

Crystallization and phase behavior of fatty acid esters of 1,3-propanediol I: pure systems

Six pure fatty acid esters of 1,3-propanediol (PADE) molecules were investigated. A careful analysis of XRD, DSC as well as SFC results has allowed the determination of their structure and phase behavior. Two beta polymorphs were observed for C10-C18 and three beta polymorphs for C8. The same first polymorph (beta1) was observed for all the samples. The second polymorph (beta2) observed for C12-C18 was different from the second beta-form observed for C8 and C10. For all properties, the short chain length C8 and C10 samples were distinguished from the C12 to C18 samples and this explained much of the observed trends in behavior. Their lamellar packing was similar and has been explained by a simple addition of multiples of the length of a carbon bond to a primitive structure. The estimated long-range order highlighted a geometric effect that enabled the small chain molecules to better order than the longest molecules. The XRD results have been confirmed by DSC. The difference in property between the short and long chain molecules has also been clearly verified by the evolution of the energy of activation for nucleation as well as the enthalpy of melting and confirmed by microscopy measurements. For all the samples, the hardness which increased with increasing chain length is correlated with final %SFC. Avrami analysis of SFC versus time indicated heterogeneous nucleation and spherulitic crystal development from sporadic nuclei, and suggested that the rate of nucleation was higher for longer chain molecules.     

A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.     

Fluorescence analysis of receptor-G protein interactions in cell membranes

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.     

Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress

Localization and staining features of the oxidant-sensitive fluorescent probe 2'7'-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.     

Monoterpenoid indole alkaloids from Palicourea crocea

A new monoterpenoid indole alkaloid N-oxide, 3,4-dihydro-1-(1-β-d-glucopyranosyloxy-1,4a,5,7a-tetrahydro-4-methoxycarbonylcyclopenta[c]pyran-7-yl)-β-carboline-N²-oxide (3) and two known monoterpenoid indole alkaloids, croceaine A (1) and psychollatine (2), were isolated from Palicourea crocea (Rubiaceae) from Trinidad. The structures of 1–3 were determined on the basis of spectral and other physical data. Compounds 1 and 2 were previously isolated from plants of different genera viz. Palicourea crocea and Psychotria umbellata, respectively, both collected in Brazil. The results support the proposal that the genus Palicourea and the subgenus Heteropsychotria should be combined into a single genus.     

Relaxational contributions to protein compressibility from ultrasonic data

The ultrasonic absorption spectra of proteins in solution generally show relaxational behaviour. There will be a corresponding dispersion of sound velocities accompanying each relaxation. The compressibility of the protein as measured by sound velocity techniques will therefore include a relaxational contribution. We have evaluated this contribution for a number of proteins and found that in some cases the relaxational contribution is a significant fraction of the total compressibility. The relaxational contribution will be large only if the molecule has a large number of degrees of freedom with low force constants. However, such motions are likely to be those involved in the biological functioning of the molecule. Care is needed in interpreting the relaxation spectrum since proton transfer processes give large apparent relaxational compressibilities.