Mechanisms responsible for surfactant changes in sepsis-induced lung injury.

Pulmonary surfactant is altered in sepsis, and these changes contribute to the predisposition of septic lungs to subsequent insults, ultimately leading to acute lung injury. Specifically, the total amount of surfactant is lower in sepsis, mainly due to decreased small aggregate (SA) surfactant pools. The amount of large aggregate (LA) surfactant is not altered. To evaluate the mechanisms responsible for these alterations, trace doses of tritium-labelled dipalmitoylphosphatidylcholine (3H-DPPC)-labelled LA were instilled intratracheally into adult rats 20 hrs after caecal ligation and perforation (CLP) or sham surgery. Animals were sacrificed at 0, 1 and 4 h after instillation and recovery of 3H-DPPC in alveolar macrophages (AM), LA and SA was measured. In separate in vitro experiments, AM isolated from CLP/sham rats were incubated with LA or SA isolated from normal animals to evaluate the uptake of these aggregates into the AM. Results showed increased surfactant radioactivity associated with AM of CLP animals compared with sham animals both in vivo and in vitro. In addition, more 3H-DPPC label remained in LA forms in the CLP animals in vivo compared with sham. These findings indicate that differences in surfactant aggregate uptake and large aggregate conversion occur in septic lungs, resulting in changes in surfactant pools.

Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.

Surfactant treatment for ventilation-induced lung injury in rats: effects on lung compliance and cytokines.

The objective of this study was to determine if exogenous surfactant therapy could prevent the harmful effects of ventilation at high tidal volumes without positive end-expiratory pressure (PEEP). Rats were randomized to either a nontreated control group (8 mL/kg 4 cm H2O PEEP), a nontreated injuriously ventilated group (20 mL/kg 0 cm H2O PEEP) or a treatment group of either 50 mg/kg, 50 mg/kg + 5% surfactant-associated protein A, 100 mg/kg exogenous surfactant followed by injurious ventilation. Isolated lungs from animals in all 5 groups were ventilated in a humidified box at 37 degrees C for 2 hours. Pressure-volume curves and light microscopy showed that surfactant treatment reduced the ventilation-induced lung injury (VILI). Inflammatory cytokines (tumor necrosis factor-alpha [TNFalpha], interleukin [IL]-1beta, and IL-6) in the lavage were significantly higher in injuriously ventilated lungs compared to the control group. However the 3 treatment groups had cytokine concentrations that were similar to the injuriously ventilated group. We conclude that surfactant treatment is beneficial in preventing VILI; however, it does not prevent the release of inrflammatory cytokines during mechanical ventilation.

Mechanical ventilation of isolated septic rat lungs: effects on surfactant and inflammatory cytokines.

The effects of mechanical ventilation (MV) on the surfactant system and cytokine secretion were studied in isolated septic rat lungs. At 23 h after sham surgery or induction of sepsis by cecal ligation and perforation (CLP), lungs were excised and randomized to one of three groups: 1) a nonventilated group, 2) a group subjected to 1 h of noninjurious MV (tidal volume = 10 ml/kg, positive end-expiratory pressure = 3 cmH(2)O), or 3) a group subjected to 1 h of injurious MV (tidal volume = 20 ml/kg, positive end-expiratory pressure = 0 cmH(2)O). Nonventilated sham and CLP lungs had similar compliance, normal lung morphology, surfactant, and cytokine concentrations. Injurious ventilation decreased compliance, altered surfactant, increased cytokines, and induced morphological changes compared with nonventilation in sham and CLP lungs. In these lungs, the surfactant system was similar in sham and CLP lungs; however, tumor necrosis factor-alpha and interleukin-6 levels were significantly higher in CLP lungs. We conclude that injurious ventilation altered surfactant independent of sepsis and that the CLP lungs were predisposed to the secretion of larger amounts of cytokines because of ventilation.

[The determination of lorazepam in human urine by gas chromatography/nitrogen-phosphorus detector].

A method to assay lorazepam in human urine has been developed. After addition of hydroxyethylflurazepam (internal standard) and hydrolysis with beta-glucuronidase, the lorazepam and hydroxyethylflurazepam were extracted with ethyl ether at pH 10.8. The analysis was performed on an HP-5 capillary column with nitrogen-phosphorus detector(NPD). The detection limit and recovery of analytes in urine were 5 micrograms/L and (83.4 +/- 3.1)% respectively. The method was successfully applied to urine specimens collected from healthy human volunteers who have ingested 2 mg of lorazepam. The method was sensitive enough to assay urine specimen excreted at 32 h after taking the medicine by volunteers.

The dynamic NMR structure of the T psi C-loop: implications for the specificity of tRNA methylation.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependent methylation of uridine-54 in the T psi C-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried and the question arises as to how RUMT gains access to the methylation site. A 17-mer RNA hairpin consisting of nucleotides 49-65 of the T psi-loop is a substrate for RUMT. Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD) methods were used to determine the solution structure of the 17-mer T-arm fragment. The loop of the hairpin exhibits enhanced flexibility which renders the conventional NMR average structure less useful compared to the more commonly found situation where a molecule exists in predominantly one major conformation. However, when resorting to softer refinement methods such as MD with time-averaged restraints, the conflicting restraints in the loop can be satisfied much better. The dynamic structure of the T-arm is represented as an ensemble of 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the T psi-loop in solution in conjunction with extensive binding studies of RUMT with the T psi C-loop and tRNA suggest that the specificity of the RUMT/ tRNA recognition is associated with tRNA tertiary structure elements. For the methylation, RUMT would simply have to break the tertiary interactions between the D- and T-loops, leading to a melting of the T-arm structure and making U54 available for methylation.

Ventilation strategies affect surfactant aggregate conversion in acute lung injury.

This study evaluated the effects of varying tidal volumes (VT) and positive end-expiratory pressure (PEEP) levels on surfactant aggregate conversion and lung function in an animal model of lung injury induced by N-nitroso-N-methylurethane. Lung-injured adult rabbits were initially ventilated using a VT of 10 ml/kg (VT10), a respiratory rate of 30 breaths/min (RR30), and a PEEP of 3.5 cm H2O. A trace dose of radiolabeled rabbit large surfactant aggregates was instilled after the onset of ventilation, and animals were then ventilated at different ventilator settings for 1 h. Ventilation strategies involving a lower VT (VT5, RR60) resulted in significantly superior oxygenation and lower surfactant aggregate conversion rates than strategies involving a higher VT ([VT10, RR30], [VT15, RR20], p < 0.05). Increasing the PEEP level to 8.0 cm H2O improved oxygenation, but it was sustained only with a low VT (VT5, RR60), and deteriorated with a high VT (VT10, RR30). Varying VT but not PEEP levels resulted in significant changes in surfactant aggregate conversion. We conclude that increased surfactant aggregate conversion resulting from suboptimal ventilation of injured lungs may play an important role in the pathophysiology of ventilation-induced lung dysfunction in acute lung injury.

Surfactant-associated protein A is important for maintaining surfactant large-aggregate forms during surface-area cycling.

Alveolar surfactant can be separated into two major subfractions, the large surfactant aggregates (LAs) and the small surfactant aggregates (SAs). The surface-active LAs are the metabolic precursors of the inactive SAs. This conversion of LAs into SAs can be studied in vitro using a technique called surface-area cycling. We have utilized this technique to examine the effect of trypsin on aggregate conversion. Our results show that trypsin increases the conversion of LAs into SAs in a concentration- and time-dependent manner. Immunoblot analysis revealed that surfactant-associated Protein A (SP-A) was the main target of trypsin. To examine further the role of SP-A in aggregate conversion, we tested the effect of Ca2+ and mannan on this process. The absence of Ca2+ (l mM EDTA) and the presence of mannan both increased the formation of SAs. Electron microscopy revealed that highly organized multilamellar and tubular myelin structures were present in samples that converted slowly to SAs. We concluded that SP-A is important for maintaining LA forms during surface-area cycling by stabilizing tubular myelin and multilamellar structures.

Alveolar surfactant aggregate conversion in ventilated normal and injured rabbits.

Alveolar surfactant can be separated into two subtypes; large aggregates and small aggregates. Large aggregates represent the surface active form of surfactant and are the metabolic precursors of small aggregates. Previous studies examined the mechanism by which large aggregates are converted into small aggregates in vitro. We used intratracheal injection of radiolabeled large aggregates in rabbits to probe the aggregate conversion in vivo. After this injection, animals were mechanically ventilated for 60 min. After the animals were killed, the lungs were lavaged, and the percentage of radiolabel present in the small aggregate fraction was determined. Our results showed that ventilation resulted in aggregate conversion and that increases in tidal volume, but not in respiratory rate, correlated with increased conversion. Aggregate conversion in rabbits with acute lung injury correlated significantly with severity of injury. We conclude that a change in surface area (i.e., respiration) is necessary for aggregate conversion in vivo and that the ventilation strategy can affect this conversion. Furthermore, increased aggregate conversion in injured lungs might contribute to increased small-to-large aggregate ratios in these lungs compared with normal lungs.

Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs.

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5' and 3' regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5' end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5' region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3' end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3' fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5' and 3' ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea - sodium dodecyl sulphate - polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-beta-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit.

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.