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Polypeptides were isolated from human bile by extraction with chloroform/methanol, followed by reversed-phase chromatography in methanol/ethylene chloride and gel filtration in chloroform/methanol. Peptides were characterized by SDS/PAGE, sequence analysis and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. This identified haemoglobin alpha chain, ATP synthase lipid-binding protein subunit 9, an N-terminal fragment of mac25/insulin-like growth factor-binding protein 7 and an internal fragment of monocyte differentiation antigen CD14, all not described previously in bile. In addition, alpha1-antitrypsin, known in bile from previous work, was also identified. The hydrophobic character of haemoglobin alpha chain is not apparent from its amino acid sequence, but the other polypeptides all have major hydrophobic segments. These results show that several proteins are removed upon organic solvent extraction used for delipidation during the preparation of samples for proteome analysis. Several of the polypeptides found are unexpectedly present in bile, suggesting that specific excretion mechanisms may be involved.

Proinsulin C-peptide ameliorates renal and autonomic nerve function and increases skeletal muscle blood flow, oxygen uptake and glucose transport in patients with insulin-dependent diabetes mellitus. These effects have in part been ascribed to the stimulatory influence of C-peptide on Na+,K+-ATPase and endothelial nitric oxide synthase. To evaluate the capacity of C-peptide to insert into lipid bilayers and form ion channels, C-peptide secondary structure and membrane interactions were studied with circular dichroism spectroscopy and size exclusion chromatography. C-peptide is shown to lack a stable secondary structure, both when part of proinsulin and when free in aqueous solution, although the N-terminal third of the peptide exhibits an alpha-helical conformation in trifluoroethanol. Moreover, C-peptide remains disordered in the aqueous solvent in the presence of lipid vesicles, regardless of vesicle composition. In conclusion, C-peptide is unlikely to elicit physiological effects through stable conformation-dependent interactions with lipid membranes.

Pulmonary surfactant contains two hydrophobic proteins, SP-B and SP-C. With the aim of identifying synthetic SP-B and SP-C substitutes for replacement therapy of respiratory distress syndromes, we have studied two transmembrane peptides and two amphipathic peptides that are located in the plane of a phospholipid bilayer. One amphipathic peptide was designed by changing the amino acid sequence, but not the composition or size, of the 21-residue peptide KL4. This peptide, designated KL(2,3) from its spacing of nonpolar and polar residues, exhibited similar alpha-helical content as KL4 but was oriented along a phospholipid bilayer plane, in contrast to the transmembrane orientation of KL4 in the same environment. The second amphipathic peptide analyzed was succinyl-LLEKLLEWLK-amide (WMAP10). KL4 more efficiently accelerated the spreading of a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Pam2GroPCho)/phosphatidylglycerol (PtdGro)/palmitic acid (PamOH), 68:22:9 (by mass), at an air/water interface than did any of the amphipathic peptides. Similarly, KL4, but not KL(2,3), when present in an interfacial monolayer composed of Pam2GroPCho/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, 7:3 (by mass), increased lipid insertion from subphase vesicles. An SP-C analogue, SP-C(Leu), with all helical valyl residues in native SP-C replaced with Leu and the palmitoylcysteines at positions 5 and 6 replaced with Ser, but otherwise with essentially the same primary structure as the native peptide, was analyzed. SP-C(Leu) exhibited similar alpha-helical content as native SP-C and a transmembrane orientation and, in contrast to poly-valyl-containing synthetic peptides, it folds into a helical conformation after acid-induced denaturation. SP-C(Leu) accelerated the spreading of Pam2GroPCho/PtdGro/PamOH, 68:22:9 (by mass), almost identically to native SP-C, and lowered the surface tension during rapid cyclic film compressions in a pulsating bubble surfactometer to near zero and 43 mN/m at minimum and maximum bubble size, respectively. Airway instillation of 2% (by mass) SP-C(Leu) combined with Pam2GroPCho/PtdGro/PamOH in preterm rabbit fetuses improved dynamic lung compliance by about 30% compared with untreated controls.

During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer’s disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.

The 42 kDa precursor of surfactant protein B generates the 79 residue mature SP-B polypeptide, which belongs to the family of saposin-like proteins and has unique functional roles in pulmonary surfactant. From sequence comparisons it has been suggested that proSP-B, in addition to SP-B, contains two saposin-like domains, but their existence has until now not been experimentally verified. The 381 residue human proSP-B was now fused to an N-terminal poly-His tag, expressed in Escherichia coli, and purified from inclusion bodies by resolubilisation with 2.5% (w/v) SDS and, after removal of SDS, subsequent metal affinity chromatography. Recombinant proSP-B thus obtained exhibits about 35% α-helical structure in sodium phosphate buffer and is proteolytically cleaved preferentially between the three saposin-like domains. These results experimentally support that proSP contains, in addition to SP-B, two further saposin-like domains.

The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO3-, SO42-, or CF3CO2- causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl- is less efficient. A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer. The extent of alpha-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL-37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered. In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 microM, and at 13-25 microM the peptide is cytotoxic against several eukaryotic cells. In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.

Antibacterial peptides were isolated from human peripheral granulocytes of a healthy donor who had been treated with granulocyte-colony stimulating factor (G-CSF) and cortisol. Peptides were solubilized in acidified chloroform/methanol, and partitioned in chloroform/methanol/water. Water- soluble polypeptides were separated by cation-exchange and reversed-phase chromatography. Several previously characterized antibacterial polypeptides were identified; defensins 1-3, defensin 4, lysozyme, eosinophil cationic protein, and calgranulin A. In addition, several histone fragments were isolated and exhibited activity against the Gram- positive bacterium Bacillus megaterium strain Bm11. These fragments included two C-terminal fragments of histone H1A, three C-terminal fragments of histone H1D, one fragment of histone H1B, and two fragments of histone H4. The molecular masses of both histone H1A fragments, as determined by electrospray (ES) MS, were 270 Da higher than those calculated from their amino acid sequences. The two histone H1A fragments corresponded to Lys152-Lys222 (7527 +/- 1 Da) and Lys167-Lys222 (6023 +/- 1 Da). Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa fragments indicated that the post-translational modification is on Lys222, the epsilon-amino group of which was conjugated with the alpha-carboxyl group of the tripeptide Arg-Gly-Gly. This finding was substantiated by digestion of the 7.5-kDa polypeptide with trypsin and analysis of the resulting peptides by ES MS and MS/MS. The tripeptide Arg-Gly-Gly corresponded uniquely to the three C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A.

BACKGROUND: Overweight and obesity in preschool children have increased worldwide in the past two to three decades. Child Health Centers provide a key setting for monitoring growth in preschool children and preventing childhood obesity. METHODS: We conducted semi-structured interviews with 15 nurses working at Child Health Centers in southwest Sweden in 2011 and 2012. All interviews were tape recorded and transcribed verbatim and imported to QSR N'Vivo 9 software. Data were analyzed deductively according to predefined themes using content analysis. RESULTS: Findings resulted in 332 codes, 16 subthemes and six main themes. The subthemes identified and described barriers and facilitators for the prevention of childhood obesity at Child Health Centers. Main themes included assessment of child's weight status, the initiative, a sensitive topic, parental responses, actions and lifestyle patterns. Although a body mass index (BMI) chart facilitated greater recognition of a child's deviant weight status than the traditional weight-for-height chart, nurses used it inconsistently. Obesity was a sensitive topic. For the most part, nurses initiated discussions of a child's overweight or obesity. CONCLUSION: CHCs in Sweden provide a favorable opportunity to prevent childhood obesity because of a systematic organization, which by default conducts growth measurements at all health visits. The BMI chart yields greater recognition of overweight and obesity in children and facilitates prevention of obesity. In addition, visualization and explanation of the BMI chart helps nurses as they communicate with parents about a child's weight status. On the other hand, inconsistent use and lack of quality assurance regarding the recommended BMI chart was a barrier to prevention, possibly delaying identification of overweight or obesity. Other barriers included emotional difficulties in raising the issue of obesity because it was perceived as a sensitive topic. Some parents deliberately wanted overweight children, which was another specific barrier. Concerned parents who took the initiative or responded positively to the information about obesity facilitated prevention activities.

BackgroundDuring the recent decade “Lean production” has become a prevalent rationalization methodology in healthcare. A commonly applied Lean tool is Value Stream Mapping (VSM). It is a participatory tool, i.a. used to identify non-Value-Adding-Work (non-VAW) in patient flows. The process results in an Action Plan suggesting interventions aiming at minimizing non-VAW in order to increase the proportion of value creation. Scientific evidence indicates that non-VAW often represents periods of physical and mental recovery. Reduction of nonVAW may therefore cause ”Work intensification”. On this background the VSM tool has been complemented by an ergonomic module (ErgoVSM) to be used in the healthcare sector(Jarebrant et al., 2010). The aim of the present study was to investigate differences in Action Plans regarding expected impact on ergonomics and performance issues when using ErgoVSM rather than VSM. Material and MethodsFourteen hospital wards were investigated, six in Denmark, two in Iceland and six in Sweden (one VSM ward refused to complete). In each country half the wards used VSM according to their ordinary Lean routines and the other half used ErgoVSM. All action plans were collected and each proposal was analysed based on triangulations between different stakeholder assessments. Data were analysed using Fisher's exact test of contingency tables of impact on four factors: Work Environment (WE, +/0/-), task/job content/system and efficiency according to VSM/ErgoVSM. ResultsOf a total of 175 proposals from all the investigated wards 106 were assessed as causing WE+, 8 WE-, 20 WE0, and 41 Not Assessable. Of the 106 WE+ proposals 78% aimed at system level changes (job content and work situation), the remaining at task level changes or not assessable. This is in contrast to the intervention proposals generally investigated in the ergonomic intervention literature focusing almost exclusively interventions at task and individual level. Using ErgoVSM rather than VSM in Sweden and Iceland resulted in a higher proportion of proposals aiming at reduction of ergonomic risks compared to the Danish wards (p=0.02). Using ErgoVSM did not result in fewer proposals aiming at improving efficiency (p=0.5).ConclusionsIntegration of ergonomic criteria into the rationalization tool VSM seems to imply a potential for more comprehensive ergonomic improvements without inhibiting the development of proposals improving efficiency. However, this impact of ErgoVSM seems to be modified by contextual factors.

We have studied a respiratory distress syndrome (RDS) occurring in newborn calves of the Belgian White and Blue (BWB) breed that represents the large majority of beef cattle in Belgium. Pulmonary surfactant isolated from 14 BWB newborn calves that died from RDS and from 7 healthy controls was analysed for composition and surface activity. An extremely low content or, in some instances, an absence of surfactant protein C (SP-C) was detected in the RDS samples by Western blotting and differential amino acid analysis [0.03±0.01% (w/w) relative to total phospholipids, compared with 0.39±0.06% for healthy controls (means±S.E.M., P < 0.001)]. The contents of surfactant protein B (SP-B) were similar in RDS and control samples. The crude surfactant samples isolated from RDS calves had higher ratios of total protein to total phospholipid, altered phospholipid profiles and lower SP-A contents. Both crude and organic extracts of RDS surfactant samples showed increased dynamic surface tension compared with healthy controls when evaluated with a pulsating-bubble surfactometer. The addition of purified SP-C to organic extracts of RDS surfactant samples lowered surface tension. Strongly decreased levels of mature SP-C associated with fatal RDS and altered surface activity in vitro have, to the best of our knowledge, not been previously reported. The mechanisms underlying RDS and the decrease in SP-C in BWB calves remain to be established.

OBJECTIVES: To investigate if, in a healthy randomly-selected population of 35-year-old men and women, there already is a relation between decreased heart rate variability and conventional risk factors for cardiovascular disease. BACKGROUND: Analysis of heart rate variability (HRV) has been used for estimating tonic autonomic activity. HRV is reduced in patients with various cardiovascular diseases. If a decreased HRV is a late phenomenon in the progression of cardiovascular disease, or if it parallels or even precedes manifest disease is unknown. DESIGN: Spectral analysis of HRV was made from 24-hour ambulatory electrocardiograms in a randomised population of healthy 35-year-old men (n = 63) and women (n = 70). The different spectral indices of HRV were analysed against gender, leucocyte count (previously described as an independent risk factor for cardiovascular disease), blood lipoproteins, smoking, heredity, body mass index (BMI) and systolic blood pressure. SETTING: A research centre of general medicine and a university hospital. RESULTS: Mean heart rate was lower, total power (TP), very low frequency power (VLF) and low frequency power (LF) were higher in men than in women. In women TP, VLF and LF were negatively correlated to BMI, smoking, triglycerides and positively correlated to HDL cholesterol. TP and VLF were also negatively correlated to risk factor score. High frequency power (HF), a marker of parasympathetic activity, was positively related to HDL cholesterol. In men, at daytime, TP, VLF, LF and HF were negatively correlated to leucocyte count. TP, VLF and LF were also negatively correlated to triglycerides and VLF also to risk factor score. CONCLUSIONS: There are correlations between HRV and known risk factors for cardiovascular disease already in a healthy 35-year-old population. A novel observation is the relationship in men between leucocyte count and heart rate variability.

The low-molecular-mass surfactant protein fraction, soluble in chloroform/methanol, contains at least two separate polypeptide chains. The 8-kDa form (type I) was isolated, [14C]carboxymethylated after reduction, and submitted to structural analysis. Its highly hydrophobic nature complicated purification, proteolytic cleavages, and sequence analysis. Acid hydrolysis in 6 M HCl for 7 days was necessary for release of branched-chain residues in full yield. Pepsin was the only enzyme found to cleave the surfactant protein and was used to complement peptide generation by chemical cleavage with CNBr. The primary structure deduced consists of 79 residues with 8 half-cystine residues, and a total of 39% branched-chain hydrophobic residues. However, 11 residues are charged at physiological pH, and all properties of the primary structure are not entirely outstanding in relation to those of other proteins. Hydrophobic segments, coupled with a presumably tight folding from the presence of disulfide bridges, probably explain the unusual properties and the solubility in organic solvents.

Efficient production of artificial spider silk fibers with properties that match its natural counterpart has still not been achieved. Recently, a biomimetic process for spinning recombinant spider silk proteins (spidroins) was presented, in which important molecular mechanisms involved in native spider silk spinning were recapitulated. However, drawbacks of these fibers included inferior mechanical properties and problems with low resistance to aqueous environments. In this work, we show that &ge, 5 h incubation of the fibers, in a collection bath of 500 mM NaAc and 200 mM NaCl, at pH 5 results in fibers that do not dissolve in water or phosphate buffered saline, which implies that the fibers can be used for applications that involve wet/humid conditions. Furthermore, incubation in the collection bath improved the strain at break and was associated with increased &beta, sheet content, but did not affect the fiber morphology. In summary, we present a simple way to improve artificial spider silk fiber strain at break and resistance to aqueous solvents.