Biomedical subjects
Jan Johansson
Publications and source records attributed to Jan Johansson.
The N-terminal propeptide of lung surfactant protein C is necessary for biosynthesis and prevents unfolding of a metastable alpha-helix.
The lung surfactant-associated protein C (SP-C) consists mainly of a polyvaline alpha-helix, which is stable in a lipid membrane. However, in agreement with the predicted beta-strand conformation of a polyvaline segment, helical SP-C unfolds and transforms into beta-sheet aggregates and amyloid fibrils within a few days in aqueous organic solvents. SP-C fibril formation and aggregation have been associated with lung disease. Here, we show that in a recently isolated biosynthetic precursor of SP-C (SP-Ci), a 12 residue N-terminal propeptide locks the metastable polyvaline part in a helical conformation. The SP-Ci helix does not aggregate or unfold during several weeks of incubation, as judged by hydrogen/deuterium exchange and mass spectrometry. Hydrogen/deuterium exchange experiments further indicate that the propeptide reduces exchange in parts corresponding to mature SP-C. Finally, in an acidic environment, SP-Ci unfolds and aggregates into amyloid fibrils like SP-C. These data suggest a direct role of the N-terminal propeptide in SP-C biosynthesis.
Folding into a beta-hairpin can prevent amyloid fibril formation.
The tetrapeptide KFFE is one of the shortest amyloid fibril-forming peptides described. Herein, we have investigated how the structural environment of this motif affects polymerization. Using a turn motif (YNGK) or a less rigid sequence (AAAK) to fuse two KFFE tetrapeptides, we show by several biophysical methods that the amyloidogenic properties are strongly dependent on the structural environment. The dodecapeptide KFFEAAAKKFFE forms abundant thick fibril bundles. Freshly dissolved KFFEAAAKKFFE is monomeric and shows mainly disordered secondary structure, as evidenced by circular dichroism, NMR spectroscopy, hydrogen/deuterium exchange measurements, and molecular modeling studies. In sharp contrast, the dodecapeptide KFFEYNGKKFFE does not form fibrils but folds into a stable beta-hairpin. This structure can oligomerize into a stable 12-mer and multiples thereof, as shown by size exclusion chromatography, sedimentation analysis, and electrospray mass spectrometry. These data indicate that the structural context in which a potential fibril forming sequence is present can prevent fibril formation by favoring self-limiting oligomerization over polymerization.
N-terminally extended surfactant protein (SP) C isolated from SP-B-deficient children has reduced surface activity and inhibited lipopolysaccharide binding.
In both humans and mice, a deficiency of surfactant protein B (SP-B) is associated with a decreased concentration of mature SP-C and accumulation of a larger SP-C peptide, denoted SP-C(i), which is not observed under normal conditions. Isolation of hydrophobic polypeptides from the lungs of children who died with two different SP-B mutations yielded pure SP-C(i) and showed only trace amounts of mature SP-C. Determination of the SP-C(i) covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35-residue segment that is identical to mature SP-C. The SP-C(i) structure determined herein is similar to that of a proposed late intermediate in the processing of proSP-C, suggesting that SP-C(i) is the immediate precursor of SP-C. In bronchoalveolar lavage fluid from transgenic mice with a focal deficiency of SP-B, SP-C(i) was detected in the biophysically active, large aggregate fraction and was associated with membrane structures that are typical for a large aggregate surfactant. However, unlike SP-C, SP-C(i) exhibited a very poor ability to promote phospholipid adsorption, gave high surface tension during cyclic film compression, and did not bind lipopolysaccharide in vitro. SP-C(i) is thus capable of associating with surfactant lipids, but its N-terminal dodecapeptide segment must be proteolytically removed to generate a biologically functional peptide. The results of this study indicate that the early postnatal fatal respiratory distress seen in SP-B-deficient children is combined with the near absence of active variants of SP-C.
Processing of pulmonary surfactant protein B by napsin and cathepsin H.
Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.
En bloc vs transhiatal esophagectomy for stage T3 N1 adenocarcinoma of the distal esophagus.
HYPOTHESIS: En bloc esophagectomy (EBE) provides improved survival over transhiatal esophagectomy (THE) in patients with similarly sized transmural tumors (T3) and lymph node metastases (N1). DESIGN: A retrospective case-control study of 2 methods of esophageal resection for cancer. SETTING: University hospital (tertiary referral center for esophageal disease). PATIENTS: There were 49 patients (27 who underwent EBE and 22 who underwent THE) with similar T3 N1 disease and the following matched criteria: tumors of similar size and location, more than 20 lymph nodes in the surgical specimen, R0 resection, no previous chemotherapy or radiation therapy, and follow-up until death or for a minimum of 5 years. Main Outcome Measure Survival adjusted for differences in demographic and patient characteristics. RESULTS: The number of nodes harvested was greatest after EBE vs THE (median, 52 vs 29 [range, 21-85 vs 20-60]; P<.001). The median number of involved nodes was similar after EBE vs THE (median, 5 vs 7 [range, 1-19 vs 1-16]). The only 2 independent factors that affected survival in a Cox analysis were the number of involved lymph nodes (P =.01) and the type of resection (P =.03). Patients who underwent EBE had a survival benefit over those who underwent THE (P =.01). The survival benefit of EBE was seen only in patients with fewer than 9 involved lymph nodes (P<.001). CONCLUSION: En bloc esophagectomy confers a better survival than THE in patients with T3 N1 disease and fewer than 9 lymph node metastases.
The antimicrobial peptide LL-37 binds to the human plasma protein apolipoprotein A-I.
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Prevalence and risk factors for ischemia, leak, and stricture of esophageal anastomosis: gastric pull-up versus colon interposition.
BACKGROUND: Reports of esophageal anastomotic complications often involve more gastric than colonic reconstructions and are incomplete because of fragmented followup by physicians unfamiliar with the surgical procedure. STUDY DESIGN: Three hundred ninety-three consecutive esophagectomy patients had prevalence and risk factors determined for graft ischemia and anastomotic leak; 363 of these patients followed for more than 1 month (median 15 months) had prevalence and risk factors determined for anastomotic stricture. RESULTS: Conduit ischemia occurred in 36 (9.2%) and anastomotic leak in 43 patients (10.9%). Risk factor for ischemia was comorbid conditions requiring therapy (Odds ratio [OR]: 2.2 [95% CI 1.1-4.3]), and for leak were ischemia (OR: 5.5 [95% CI 2.5-12.1]), neoadjuvant therapy (OR: 2.2 [95% CI 1.1-4.5]), and comorbid conditions (OR: 2.1 [95% CI 1.1-3.9]). A stricture developed in 80 patients (22.0%). Risk factors were ischemia (OR: 4.4 [95% CI 2.0-9.6]), anastomotic leak (OR: 3.8 [95% CI 1.9-7.6]), and increasing preoperative weight (p = 0.022). The prevalence of ischemia was similar after gastric (10.4%) versus colonic (7.4%) reconstruction; leak and stricture were more common (14.3% versus 6.1%, p = 0.013, 31.3% versus 8.7%, p < 0.0001, respectively) and strictures were more severe (11.2% versus 2%, p = 0.001) after gastric pull-up. Patients free of ischemia and leak who developed stricture were more likely to have had a gastric pull-up (25% versus 7%, p < 0.0001). Dilatation was effective treatment in 93% of patients. CONCLUSIONS: After esophagectomy 10% of patients will develop conduit ischemia or an anastomotic leak and 22% will develop anastomotic stricture. Anastomotic leak and strictures are more common and the strictures are more severe after gastric pull-up compared with colon interposition. Dilatation is a safe and effective treatment.
Stabilization of discordant helices in amyloid fibril-forming proteins.
Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.
Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers: implications for the epilepsy seen in the dementia FENIB.
The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with the age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive centre loop of one neuroserpin molecule with beta-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into beta-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.
Antibacterial activities of the cathelicidins prophenin (residues 62 to 79) and LL-37 in the presence of a lung surfactant preparation.
The antibacterial activities of the cathelicidin peptides LL-37 and an 18-residue C-terminal fragment of prophenin, corresponding to positions 62 to 79 of native prophenin (PF-18), were analyzed in the presence of a modified surfactant preparation isolated from minced porcine lungs. At low micromolar concentrations, both LL-37 and PF-18 showed significant activities against different serotypes of group B streptococci, with LL-37 being more active on a molar basis. The surfactant preparation at a concentration of 10 mg/ml partly blocked the antibacterial activity of 9 microM LL-37 and completely blocked the antibacterial activity of 9 microM PF-18. However, 10 mg of the surfactant preparation per ml had only minor inhibitory effects on LL-37 and PF-18 at 90 microM. Addition of up to 900 microM PF-18 did not affect the surface properties of the surfactant preparation. These data suggest that surfactant preparations containing antimicrobial peptides could be useful for the local treatment of pulmonary infections.
A synthetic surfactant based on a poly-Leu SP-C analog and phospholipids: effects on tidal volumes and lung gas volumes in ventilated immature newborn rabbits.
Available surfactants for treatment of respiratory distress syndrome in newborn infants are derived from animal lungs, which limits supply and poses a danger of propagating infectious material. Poly-Val-->poly-Leu analogs of surfactant protein (SP)-C can be synthesized in large quantities and exhibit surface activity similar to SP-C. Here, activity of synthetic surfactants containing a poly-Leu SP-C analog (SP-C33) was evaluated in ventilated premature newborn rabbits. Treatment with 2.5 ml/kg body wt of 2% (wt/wt) SP-C33 in 1,2-dipalmitoyl-sn-3-glycero phosphoryl choline (DPPC)-1-palmitoyl-2-oleoyl-sn-3-glycero phosphoryl choline (POPC)-1-palmitoyl-2-oleoyl-sn-3-glycero phosphoryl glycerol (POPG), 68:0:31, 68:11:20, or 68:16:15 (wt/wt/wt) suspended at 80 mg/ml gave tidal volumes (Vt) of 20-25 ml/kg body wt, with an insufflation pressure of 25 cmH2O and no positive end-expiratory pressure (PEEP), comparable to the Vt for animals treated with the porcine surfactant Curosurf. Nontreated littermates had a Vt of approximately 2 ml/kg body wt. The Vt for SP-C33 in DPPC-egg phosphatidylglycerol-palmitic acid [68:22:9 (wt/wt/wt)], DPPC-POPG-palmitic acid [68:22:9 (wt/wt/wt)], and DPPC-POPC-POPG [6:2:2 (wt/wt/wt)] was 15-20 ml/kg body wt. Histological examination of lungs from animals treated with SP-C33-based surfactants showed incomplete, usually patchy air expansion of alveolar spaces associated with only mild airway epithelial damage. Lung gas volume after 30 min of mechanical ventilation were more than threefold larger in animals treated with Curosurf than in those receiving SP-C33 in DPPC-POPC-POPG, 68:11:20. This difference could be largely counterbalanced by ventilation with PEEP (3-4 cmH2O). An artificial surfactant based on SP-C33 improves Vt in immature newborn animals ventilated with standardized peak pressure but requires PEEP to build up adequate lung gas volumes.
Cellular antiendotoxin activities of lung surfactant protein C in lipid vesicles.
The respiratory system is continuously exposed to airborne particles containing lipopolysaccharide. Our laboratory established previously that the hydrophobic surfactant protein C (SP-C) binds to lipopolysaccharide and to one of its cellular receptors, CD14. Here we examined the influence of SP-C, and of a synthetic analog, on some cellular in vitro effects of lipopolysaccharide. When associated with vesicles of dipalmitoylphosphatidylcholine, SP-C inhibits the binding of a tritium-labeled lipopolysaccharide to the macrophage cell line RAW 264.7. Under similar conditions of presentation, SP-C inhibits the mitogenic effect of lipopolysaccharide on mouse splenocytes, and inhibits the lipopolysaccharide-induced production of tumor necrosis factor-alpha by peritoneal and alveolar macrophages, and of nitric oxide by RAW 264.7 cells. In contrast, tumor necrosis factor-alpha production induced by a lipopeptide, and nitric oxide production induced by picolinic acid, were not affected by SP-C. The lipopolysaccharide-binding capacity of SP-C is resistant to peroxynitrite, a known mediator of acute lung injury formed by reaction of nitric oxide with superoxide anions. These results indicate that SP-C may play a role in lung defense; SP-C resists degradation under inflammatory conditions and traps lipopolysaccharide, preventing it from inducing production of noxious mediators in alveolar cells.
Molecular determinants for amyloid fibril formation: lessons from lung surfactant protein C.
Amyloid fibrils are polymers composed of proteins in beta-sheet conformation, which are found in at least 20 diseases for which no cure is available. These diseases include Alzheimer's disease and spongiform encephalopathies, in which the amyloid beta-peptide and the prion protein (PrP), respectively, form amyloid. All fibrils are morphologically very similar although the native structures of the corresponding proteins are widely different. Proteins that are not known to form fibrils in vivo can do so under conditions where unfolding intermediates are well populated. This indicates that fibril formation can arise from most, if not all, polypeptide chains under certain conditions, and that nature has found ways to avoid fibrillogenic protein conformations. In support of this, it was recently found that unpaired beta-strands present in native proteins are prevented from forming intermolecular beta-sheets, by strategic placement of prolines and charged residues for example. Structural studies of the lung surfactant-associated protein C (SP-C) have revealed determinants for amyloid fibril formation. The poly-valine alpha-helix of SP-C spontaneously converts to beta-sheet aggregates in vitro and SP-C amyloid fibrils are found in pulmonary alveolar proteinosis. A beta, PrP, and SP-C harbour an alpha-helix which is strongly predicted to form a beta-strand, and in all cases investigated so far such alpha-helix/beta-sheet discordance correlates with the ability to form beta-sheet aggregates and fibrils.
Intramural delivery of recombinant apolipoprotein A-IMilano/phospholipid complex (ETC-216) inhibits in-stent stenosis in porcine coronary arteries.
BACKGROUND: We have previously demonstrated vasculoprotective effects after repeated intravenous administration of recombinant apolipoprotein A-IMilano (apoA-Im)/phospholipid complex. In this study, we sought to determine the effects of local recombinant apoA-Im/1-palmitoyl,2-oleoyl phosphatidylcholine complex (ETC-216) delivered intramurally via the Infiltrator catheter on luminal narrowing in a porcine coronary artery stent overstretch injury model. METHODS AND RESULTS: In twelve domestic swine ( approximately 25 kg), two arteries each were infiltrated with 0.4 mL ETC-216 (14 mg/mL) or vehicle control immediately before deployment of GFX stents (stent:artery ratio=1.3:1). Animals were euthanized at day 28, and evaluation by QCA revealed a significant improvement in mean lumen loss index with ETC-216 treatment (21+/-22% versus 43+/-13% lumen loss; P=0.01). Histomorphometric analysis showed that ETC-216 treatment significantly reduced the intimal area (6.7+/-1.5 versus 5.2+/-1.4 mm2, -22%; P=0.02) and the stenosis index (0.76+/-0.15 versus 0.59+/-0.15; P=0.01), and increased the lumen area (2.1+/-1.4 versus 3.7+/-1.8 mm2, +76%; P=0.02). Regression analysis showed significant differences in lumen area (P=0.004), neointimal area (P=0.003), stenosis index (P=0.001), and neointimal thickness (P=0.003) adjusted for injury score in favor of ETC-216. CONCLUSIONS: A single intramural administration of ETC-216 significantly inhibited injury-induced luminal narrowing in the porcine stent overstretch model through reduction of intimal hyperplasia. These data show that local intracoronary delivery of ETC-216 may be useful to prevent restenosis after coronary stenting.
Cervical or thoracic anastomosis after esophageal resection and gastric tube reconstruction: a prospective randomized trial comparing sutured neck anastomosis with stapled intrathoracic anastomosis.
OBJECTIVE: The purpose of the study was to compare in prospective randomized fashion a manually sutured esophagogastric anastomosis in the neck and a stapled in the chest after esophageal resection and gastric tube reconstruction. SUMMARY BACKGROUND DATA: Despite the fact that all reconstructions after esophagectomy will result in a cervical or a thoracic anastomosis, controversy still exists as to the optimal site for the anastomosis. In uncontrolled studies, both neck and chest anastomoses have been advocated. The only reported randomized study is difficult to evaluate because of varying routes of the substitute and different anastomotic techniques within the groups. The reported high failure rate of stapled anastomoses in the neck and the fact that most surgeons prefer to suture cervical anastomoses made us choose this technique for anastomosis in the neck. Our routine and the preference of most surgeons to staple high thoracic anastomoses became decisive for type of thoracic anastomoses. METHODS: Between May 9, 1990 and February 5, 1996, 83 patients undergoing esophageal resection were prospectively randomized to receive an esophagogastric anastomosis in the neck (41 patients) or an esophagogastric anastomosis in the chest (42 patients). To evaluate selection bias, patients undergoing esophageal resection during the same period but not randomized (n = 29) were also followed and compared with those in the study (n = 83). Objective measurements of anastomotic level and diameter were assessed with an endoscope and balloon catheter 3, 6, and 12 months after surgery. The long-term survival rates were compared with the log-rank test. RESULTS: Two patients (1.8%) died in hospital, and the remaining 110 patients were followed until death or for a minimum of 60 months. The genuine 5-year survival rate was 29% for chest anastomoses and 30% for neck anastomoses. The overall leakage rate was 1.8% (2 cases of 112) with no relation to mortality or anastomotic method. All patients in the randomized group had tumor-free proximal and distal resection lines, but 1 patient in the nonrandomized group had tumor infiltrates in the proximal resection margin. At 3, 6, and 12 months after operation, there was no difference in anastomotic diameter between the esophagogastric anastomosis in the neck and in the thorax (P = 0.771), and both increased with time (P = 0.004, ANOVA repeated measures). Body weight development was the same in the two groups. With similar results in randomized and nonrandomized patients, study bias was eliminated. CONCLUSIONS: When performed in a standardized way, neck and chest anastomoses after esophageal resection are equally safe. The additional esophageal resection of 5 cm in the neck group did not increase tumor removal and survival; on the other hand, it did not adversely influence morbidity, anastomotic diameter, or eating as reflected by body weight development.
Interaction of pulmonary surfactant protein C with CD14 and lipopolysaccharide.
In addition to their effects on alveolar surface tension, some components of lung surfactant also have immunological functions. We found recently that the hydrophobic lung surfactant protein SP-C specifically binds to the lipid A region of lipopolysaccharide (LPS). In this study, we show that SP-C also interacts with CD14. Four observations showed cross talk between the three molecules SP-C, LPS, and CD14. (i) Like LBP, SP-C allows the binding of a fluorescent LPS to cells expressing CD14 (the other surfactant components were ineffective). (ii) Recombinant radiolabeled CD14 and SP-C (or a synthetic analog of SP-C) interact in a dose-dependent manner. (iii) LPS blocks the binding of radiolabeled CD14 to SP-C-coated wells. (iv) SP-C enhances the binding of radiolabeled CD14 to LPS-coated wells. These results, obtained with native murine SP-C and with three synthetic analogs, suggest that LPS and CD14 interact with the same region of SP-C and that binding of SP-C modifies the conformation of CD14 or the accessibility of its LPS-binding site, allowing it to bind LPS. This ability of SP-C to interact with the pattern recognition molecule CD14 extends the possible immunological targets of SP-C to a large panel of microorganisms that can enter the airways.
Surfactant lavage demonstrates protein fibrils in a neonate with congenital surfactant protein b deficiency.
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