Protection of lung tissue against ischemia/reperfusion injury by preconditioning with inhaled nitric oxide in an in situ pig model of normothermic pulmonary ischemia

Topical administration of nitric oxide (NO) by inhalation is currently used as therapy in various pulmonary diseases, but preconditioning with NO to ameliorate lung ischemia/reperfusion (I/R) injury has not been fully evaluated. In this study, we investigated the effects of NO inhalation on functional pulmonary parameters using an in situ porcine model of normothermic pulmonary ischemia. After left lateral thoracotomy, left lung ischemia was maintained for 90 min, followed by a 5h reperfusion period (group I, n = 7). In group II (n = 6), I/R was preceded by inhalation of NO (10 min, 15 ppm). Animals in group III (n = 7) underwent sham surgery without NO inhalation or ischemia. In order to evaluate the effects of NO preconditioning, lung functional and hemodynamic parameters were measured, and the zymosan-stimulated release of reactive oxygen species in arterial blood was determined. Animals in group I developed significant pulmonary I/R injury, including pulmonary hypertension, a decreased pO(2) level in pulmonary venous blood of the ischemic lung, and a significant increase of the stimulated release of reactive oxygen species. All these effects were prevented, or the onset (release of reactive oxygen species) was delayed, by NO inhalation. These results indicate that preconditioning by NO inhalation before lung ischemia is protective against I/R injury in the porcine lung.     

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Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.     

Influence of wheat and rye parents on agronomic characters in primary hexaploid and octoploid triticale

Summary Thirty-five hexaploid and twenty octoploid primary triticales (xTriticosecale Wittmack) derived from homozygous wheat and rye inbred lines were used (1) to investigate the parental wheat, rye, and interaction effects and (2) to estimate quantitative genetic parameters for agronomic traits. The winter triticales were tested in four environments in a three-replicate split-plot design with drilled 1 m² plots. Superior performance of hexaploid triticales as compared to the octoploids was revealed. Substantial genetic variation and high heritability estimates were found for nearly all of the characters investigated. Estimates of wheat, rye, and wheat×rye interaction variance components disclosed parental main effects to be the most important source of genetic variation in primary triticales. The rye parent was dominant for all characters affecting fertility, and the wheat parent was more important for vegetative development. Character correlations were very similar for triticales of both ploidy levels. The lack of association between grain yield and tillering and the positive correlation between kernels per spike and thousand kernel weight indicated physiological disorders specific for primary triticales.     

Identification of a neutralizing epitope on glycoprotein gp58 of human cytomegalovirus.

Human cytomegalovirus contains an envelope glycoprotein of 58 kilodaltons (gp58). The protein, which is derived from a glycosylated precursor molecule of 160 kilodaltons via proteolytic cleavage, is capable of inducing neutralizing antibodies. We have mapped the epitopes recognized by the neutralizing monoclonal antibody 7-17 and a second antibody (27-287) which is not neutralizing. Overlapping fragments of the carboxy-terminal part of the open reading frame coding for gp58 were expressed in Escherichia coli as beta-galactosidase fusion proteins. The reactivities of antibodies 7-17 and 27-287 were determined by Western blot (immunoblot) analysis. Both antibodies recognized sequences between amino acids 608 and 625 of the primary gp58 translation product. The antibodies almost completely inhibited one another in a competitive binding assay with intact virus as antigen. Moreover, antibody 27-287 was able to inhibit the complement-independent neutralizing activity of antibody 7-17.     

Osteosarcomagenic doses of radium (224Ra) and infectious endogenous retroviruses enhance proliferation and osteogenic differentiation of skeletal tissue differentiating in vitro

Cartilage tissue from embryonic mice which undergoes osteogenic differentiation during in vitro cultivation was used to study the effect of osteosarcomagenic doses of -irradiation and bone-tumor-inducing retroviruses on proliferation and phenotypic differentiation of skeletal cells in a defined tissue culture model. Irradiated mandibular condyles showed dose-dependent enhancement of cell proliferation at day 7 of the culture and increased osteogenic differentiation at day 14. Maximal effects were found with 7.4 Bq/ml of²²⁴Ra-labeled medium. Doses of 740 and 7400 Bq/ml of²²⁴Ra-labeled medium induced increasing cell death. Retrovirus infection enhanced osteogenic differentiation and extended the viability of irradiated cells. After transplantation none of the treated tissues developed tumors in syngeneic mice.     

In Vitro and In Vivo Activity of Hamycin Against Blastomyces dermatitidis

Clinical responses of patients with blastomycosis to treatment with hamycin have been variable. An explanation for this was sought in a series of studies in which in vitro and in vivo susceptibilities to hamycin of five strains of Blastomyces dermatitidis were compared. Minimal inhibitory concentrations of hamycin for the five strains indicated uniformly high levels of in vitro susceptibility (0.008 to 0.016 μg/ml). In vivo activity was measured in infected mice treated intraperitoneally for a period of 28 days with doses of the drug ranging from 0.001 to 0.030 mg per mouse. Significant differences in response to treatment among the five strains were noted (P < 0.001), and protective doses were found to vary from 0.001 to >0.030 mg per mouse per day. Further observations of infected mice after treatment revealed marked rates of relapsing infection, and several strains caused death. Persistent inapparent infections were also detected on culture of selected organs. Toxicity due to hamycin alone was not observed. These results suggest that variations in clinical responses to hamycin therapy in treatment of blastomycosis reflect differences in pathogenesis and host response in vivo to the infecting organism rather than differences in susceptibility of B. dermatitidis to hamycin.     

Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3‐type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, such as respiratory heme‐copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In this study, we analyzed Cu delivery to the cbb₃‐type cytochrome c oxidase (cbb₃‐Cox) of Rhodobacter capsulatus. We identified the PCu_AC‐like periplasmic chaperone PccA and analyzed its contribution to cbb₃‐Cox assembly. Our data demonstrate that PccA is a Cu‐binding protein with a preference for Cu(I), which is required for efficient cbb₃‐Cox assembly, in particular, at low Cu concentrations. By using in vivo and in vitro cross‐linking, we show that PccA forms a complex with the Sco1‐homologue SenC. This complex is stabilized in the absence of the cbb₃‐Cox‐specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. These data demonstrate that the interplay between PccA and SenC not only is required for Cu delivery during cbb₃‐Cox assembly but also regulates Cu homeostasis in R. capsulatus.     

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