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 HCO 3 ؊ , SO 4 2؊ , or CF 3 CO 2 ؊ 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 ␣-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 M, and at 13-25 M 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.

Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.

The dominating functional property of pulmonary surfactant is to reduce the surface tension at the alveolar aidliquid interface, and thereby prevent the lungs from collapsing at the end of expiration. In addition, the system exhibits host-defense properties. Insufficient amounts of pulmonary surfactant in premature infants causes respiratory distress syndrome, a serious threat which nowadays can be effectively treated by airway instillation of surfactant preparations. Surfactant is a mixture of many molecular species, mainly phospholipids and specific proteins, surfactant protein A (SP-A), SP-B, SP-C and SP-D. SP-A and SP-D are water-soluble and belong to the collectins, a family of large multimeric proteins which structurally exhibit collagenousAectin hybrid properties and functionally are Caz+-dependent carbohydrate binding proteins involved in innate host-defence functions. SP-A and SP-D also bind lipids and SP-A is involved in organization of alveolar surfactant phospholipids. SP-B belongs to another family of proteins, which includes also lipid-interacting polypeptides with antibacterial and lytic properties. SP-B is a 17.4-kDa homodimer and each subunit contains three intrachain disulphides and has been proposed to contain four amphipathic helices oriented pairwise in an antiparallel fashion. SP-A, SP-B and SP-D all have been detected also in the gastrointestinal tract. SP-C, in contrast, appears to be a unique protein with extreme structural and stability properties and to exist exclusively in the lungs. SP-C is a lipopeptide containing covalently linked palmitoyl chains and is folded into a 3.7-nm a-helix with a central 2.3-nm all-aliphatic part, making it perfectly suited to interact in a transmembranous way with a fluid bilayer composed of dipalmitoylglycerophosphocholine, the main component of surfactant. Homozygous genetic deficiency of proSP-B causes lethal respiratory distress soon after birth and is associated with aberrant processing of the precursor of SP-C. This review focuses on the chemical composition, structures and interactions of the pulmonary surfactant, in particular the associated proteins.

The C-peptide of proinsulin is important for the biosynthesis of insulin but has for a long time been considered to be biologically inert. Data now indicate that C-peptide in the nanomolar concentration range binds specifically to cell surfaces, probably to a G protein-coupled surface receptor, with subsequent activation of Ca(2+)-dependent intracellular signaling pathways. The association rate constant, K(ass), for C-peptide binding to endothelial cells, renal tubular cells, and fibroblasts is approximately 3. 10(9) M(-1). The binding is stereospecific, and no cross-reaction is seen with insulin, proinsulin, insulin growth factors I and II, or neuropeptide Y. C-peptide stimulates Na(+)-K(+)-ATPase and endothelial nitric oxide synthase activities. Data also indicate that C-peptide administration is accompanied by augmented blood flow in skeletal muscle and skin, diminished glomerular hyperfiltration, reduced urinary albumin excretion, and improved nerve function, all in patients with type 1 diabetes who lack C-peptide, but not in healthy subjects. The possibility exists that C-peptide replacement, together with insulin administration, may prevent the development or retard the progression of long-term complications in type 1 diabetes.

The nuclear magnetic resonance (NMR) structure of the pulmonary surfactant-associated lipoplypeptide C (SP-C) was determined in a mixed solvent of C2H3Cl/C2H3OH/ 1 M HCl 32:64:5 (v/v). Sequence-specific 1H NMR assignments and the collection of conformational constraints were achieved with two-dimensional 1H NMR, and the structure was calculated with the distance geometry program DIANA. The root mean square deviations for the well-defined polypeptide segment of residues 9-34 calculated for the 20 best energy-minimized DIANA conformers relative to their mean are 0.5 and 1.3 A for the polypeptide backbone atoms N, C alpha, and C', and for all heavy atoms, respectively. The 35-residue polypeptide chain of SP-C forms an alpha-helix between positions 9 and 34, which includes two segments of seven and four consecutive valyls that are separated by a single leucyl residue. The N-terminal hexapeptide segment, which includes two palmitoylcysteinyls, is flexibly disordered. The length of the alpha-helix is about 37 A, and the helical segment of residues 13-28, which contains exclusively aliphatic residues with branched side chains, is 23-A long and about 10 A in diameter. The alpha-helix is outstandingly regular, with virtually identical chi 1 angles for all valyl residues. The observation of a helical structure of SP-C was rather unexpected, considering that Val is generally underrepresented in alpha-helices, and it provides intriguing novel insights into the structural basis of SP-C functions as well as into general structural aspects of protein-lipid interactions in biological membranes.

Recombinant A-I(Milano)/PC prevented progression of aortic atherosclerosis and reduced lipid and macrophage content of plaques in apo E-deficient mice despite severe hypercholesterolemia. Thus, A-I(Milano)/PC may have a role in inhibiting progression and promoting stabilization of atherosclerosis.

The secondary structure of native and depalmitoylated porcine surfactant-associated protein C (SP-C) was studied by attenuated total reflection Fourier-transform infrared spectroscopy. Both forms of porcine SP-C adopt mainly an alpha-helical conformation. These two forms of the protein were reconstituted in a lipid bilayer. The insertion of the protein in a membrane is associated with an increase of the alpha-helical content. Dichroic measurements show that, in both cases, the long axis of the alpha-helix is oriented parallel to the lipid acyl chains.

Background-Carriers of the apolipoprotein A-I Milano (apoA-I M ) mutant present with very low plasma HDL cholesterol and moderate hypertriglyceridemia, apparently not leading to premature coronary heart disease. The objective of this study was to establish whether this high-risk lipid/lipoprotein profile is associated with structural changes in the carotid arteries and heart, indicative of preclinical atherosclerosis. Methods and Results-Twenty-one A-I M carriers were compared with age-and sex-matched control subjects from the same kindred and with 2 series of matched subjects with primary hypoalphalipoproteinemia (HA). Structural changes in the carotid arteries were defined as the intima-media thickness (IMT) measured by B-mode ultrasound. HA subjects, both recruited among patients attending our Lipid Clinic and blood donors, showed significant thickening of the carotids (average IMT, 0.86Ϯ0.25 and 0.88Ϯ0.29 mm, respectively) compared with control subjects (average IMT, 0.64Ϯ0.12 mm); the apoA-I M carriers instead showed normal arterial thickness (average IMT, 0.63Ϯ0.10 mm). Moreover, a significantly higher prevalence of atherosclerotic plaques was found in patients and blood donors with HA (both 57%) compared with apoA-I M carriers (33%) and control subjects (21%). Echocardiographic findings and maximal treadmill ECG did not differ significantly between apoA-I M carriers and control subjects, apart from a slight increase in left ventricular end-diastolic dimension in the carriers. Conclusions-Despite severe HA, carriers of the apoA-I M mutant do not show structural changes in the arteries and heart, in contrast to HA subjects, who are characterized by a marked increase in carotid IMT and increased prevalence of atherosclerotic plaques.