Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes. Aib, α-aminoisobutyric acid; Z, benzyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; Ac, acetyl; CTC, chlortetracycline.

Fluorescent alamethicin fragments A study of membrane activity and aqueous phase aggregation

The linear polypeptide antibiotic alamethicin is known to form channels in artificial lipid membranes. Synthetic 13- and 17-residue alamethicin fragments, labelled with a fluorescent dansyl group at the N-terminus, have been shown to translocate divalent cations across phospholipid membranes and to uncouple oxidative phosphorylation in rat liver mitochondria, in a manner analogous to the parent peptides. From studies of the aqueous phase aggregation behavior of the peptides, as well as their interaction with rat liver mitochondria, it is concluded that the interaction of the peptides with membranes is a complex process, probably involving both aqueous and membrane phase aggregation.

Infrared studies on the conformation of synthetic alamethicin fragments and model peptides containing .alpha.-aminoisobutyric acid

ABSTRACT: Infrared studies of synthetic alamethicin fragments and model peptides containing a-aminoisobutyric acid (Aib) have been carried out in solution. Tripeptides and larger fragments exhibit a strong tendency to form /3 turns, stabilized by 4 - 1 10-atom hydrogen bonds. Dipeptides show less well-defined structures, though C5 and C7 conformations are detectable. Conformational restrictions imposed by Aib residues result in these peptides populating a limited range of states. Integrated intensities of the hydrogen-bonded N-H stretching band can be used to quantitate the number of intramolecular hydrogen bonds. Predictions made from infrared data are in excellent agreement with nuclear magnetic resonance and X-ray diffraction studies. Assignments of the urethane and tertiary amide carbonyl groups in the free state have been made in model peptides. Shifts to lower frequency on hydrogen bonding are observed for the carbonyl groups. The 1-6 segment of alamethicin is shown to adopt a 310 helical structure stabilized by four intramolecular hydrogen bonds. The fragments Boc-Leu-Aib-Pro-Val-Aib-OMe (1 2-1 6) and Boc-Gly-Leu-Aib-Pro-Val-Aib-OMe (1 1-1 6) possess structures involving 4 - 1 and 5 - 1 hydrogen bonds. Supporting evidence for these structures is obtained from proton nuclear magnetic resonance studies.

Capillary electrochromatography: a versatile instrumental technique for nanodomain interaction studies

This doctoral thesis describes the development of a miniaturized capillary electrochromatography (CEC) technique suitable for the study of interactions between various nanodomains of biological importance. The particular focus of the study was low-density lipoprotein (LDL) particles and their interaction with components of the extracellular matrix (ECM). LDL transports cholesterol to the tissues through the blood circulation, but when the LDL level becomes too high the particles begin to permeate and accumulate in the arteries. Through binding sites on apolipoprotein B-100 (apoB-100), LDL interacts with components of the ECM, such as proteoglycans (PGs) and collagen, in what is considered the key mechanism in the retention of lipoproteins and onset of atherosclerosis. Hydrolytic enzymes and oxidizing agents in the ECM may later successively degrade the LDL surface. Metabolic diseases such as diabetes may provoke damage of the ECM structure through the non-enzymatic reaction of glucose with collagen. In this work, fused silica capillaries of 50 micrometer i.d. were successfully coated with LDL and collagen, and steroids and apoB-100 peptide fragments were introduced as model compounds for interaction studies. The LDL coating was modified with copper sulphate or hydrolytic enzymes, and the interactions of steroids with the native and oxidized lipoproteins were studied. Lipids were also removed from the LDL particle coating leaving behind an apoB-100 surface for further studies. The development of collagen and collagen decorin coatings was helpful in the elucidation of the interactions of apoB-100 peptide fragments with the primary ECM component, collagen. Furthermore, the collagen I coating provided a good platform for glycation studies and for clarification of LDL interactions with native and modified collagen. All methods developed are inexpensive, requiring just small amounts of biomaterial. Moreover, the experimental conditions in CEC are easily modified, and the analyses can be carried out in a reasonable time frame. Other techniques were employed to support and complement the CEC studies. Scanning electron microscopy and atomic force microscopy provided crucial visual information about the native and modified coatings. Asymmetrical flow field-flow fractionation enabled size measurements of the modified lipoproteins. Finally, the CEC results were exploited to develop new sensor chips for a continuous flow quartz crystal microbalance technique, which provided complementary information about LDL ECM interactions. This thesis demonstrates the potential of CEC as a valuable and flexible technique for surface interaction studies. Further, CEC can serve as a novel microreactor for the in situ modification of LDL and collagen coatings. The coatings developed in this study provide useful platforms for a diversity of future investigations on biological nanodomains.

Molecular dynamics simulations of CXCL-8 and its interactions with a receptor peptide, heparin fragments, and sulfated linked cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 310-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment−CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8−receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

Promotion of fibronectin independent invasion by C5a peptidase into epithelial cells in group A Streptococcus

Background & objectives: Group A Streptococcus, causative agent of several clinical manifestations codes for multiple protein invasins which help the bacterium to enter non-phagocytic cells. C5a peptidase (SCPA) is a surface protein conserved among different serotypes of M1 strain. The present study was taken up to study SCPA promoted fibronectin independent entry of GAS into epithelial cells. Methods: An isogenic 90226 emm1DeltaAB (M1(-)) mutant was constructed, with thermosensitive pGhost vector. This isogenic M1(-) mutant expressed SCPA on the surface as determined by Western blotting and immunofluorescence. Results: On preincubation with anti-SCPA serum, the isogenic M1(-) strain exhibited 54 per cent decreased invasion as compared to the bacteria incubated with control serum. Also, purified recombinant SCPA proteins blocked internalization of M1(-) streptococci into HEp-2 cells. The M1(-) strain invaded at the same efficiency in the presence or absence of fibronectin. Interpretation & conclusion: These results suggested that SCPA acted as a potential invasin of group A streptococcus and promoted invasion independent of fibronectin.

Racemization at proline residues during peptide bond formation : A study of diastereomeric mixtures of synthetic alamethicin fragments by 270 MHz 1H NMR

The stepwise synthesis of amino terminal pentapeptide of alamethicin, Z-Aib-Pro-Aib-Ala-Aib-OMe, by the dicyclohexylcarbodiimide mediated couplings leads to extensive racemization at the Ala and Pro residues. Racemization is largely suppressed by the use of additives like N-hydroxysuccinimide and 1-hydroxybenzotriazole. The presence of diastereomeric peptides may be detected by the observation of additional methyl ester and benzylic methylene signals in the 270 MHz 1H NMR spectra. Unambiguous spectral assignment of the signals to the diastereomers has been carried out by the synthesis and NMR studies of the D-Ala tetra and pentapeptides. The racemization at Pro is of particular relevance in view of the reported lack of inversion at C-terminal Pro on carboxyl activation.

Pathobiological Aspects of Nonreheumatic Aortic Valve Stenosis

Aortic valve stenosis (AS) is an active disease process akin to atherosclerosis, with chronic inflammation, lipid accumulation, extracellular matrix remodeling, fibrosis, and extensive calcification of the valves being characteristic features of the disease. The detailed mechanisms and pathogenesis of AS are still incompletely understood, however, and pharmacological treatments targeted toward components of the disease are not currently available. In this thesis project, my coworkers and I studied stenotic aortic valves obtained from 86 patients undergoing valve replacement for clinically significant AS. Non-stenotic control valves (n=17) were obtained from patients undergoing cardiac transplantation or from organ donors without cardiac disease. We identified a novel inflammatory factor, namely mast cell, in stenotic aortic valves and present evidence showing that this multipotent inflammatory cell may participate in the pathogenesis of AS. Using immunohistochemistry and double immunofluorescence stainings, we found that a considerable number of mast cells accumulate in stenotic valves and, in contrast to normal valves, the mast cells in diseased valves were in an activated state. Moreover, valvular mast cells contained two effective proteases, chymase and cathepsin G, which may participate in adverse remodeling of the valves either by inducing fibrosis (chymase and cathepsin G) or by degrading elastin fibers in the valves (cathepsin G). As chymase and cathepsin G are both capable of generating the profibrotic peptide angiotensin II, we also studied the expression and activity of angiotensin-converting enzyme (ACE) in the valves. Using RT-PCR, imunohistochemistry, and autoradiography, we observed a significant increase in the expression and activity of ACE in stenotic valves. Besides mast cell-derived cathepsin G, aortic valves contained other elastolytic cathepsins (S, K, and V). Using immunohistochemistry, RT-PCR, and fluorometric microassay, we showed that the expression and activity of these cathepsins were augmented in stenotic valves. Furthermore, in stenotic but not in normal valves, we observed a distinctive pattern of elastin fiber degradation and disorganization. Importantly, this characteristic elastin degradation observed in diseased valves could be mimicked by adding exogenous cathepsins to control valves, which initially contained intact elastin fibers. In stenotic leaflets, the collagen/elastin ratio was increased and correlated positively with smoking, a potent AS-accelerating factor. Indeed, cigarette smoke could also directly activate cultured mast cells and fibroblasts. Next, we analyzed the expression and activity of neutral endopeptidase (NEP), which parallels the actions of ACE in degrading bradykinin (BK) and thus inactivates antifibrotic mechanisms in tissues. Real-time RT-PCR and autoradiography revealed NEP expression and activity to be enhanced in stenotic valves compared to controls. Furthermore, both BK receptors (1 and 2) were present in aortic valves and upregulated in stenotic leaflets. Isolated valve myofibroblasts expressed NEP and BK receptors, and their upregulation occurred in response to inflammation. Finally, we observed that the complement system, a source of several proinflammatory mediators and also a potential activator of valvular mast cells, was activated in stenotic valves. Moreover, receptors for the complement-derived effectors C3a and C5a were expressed in aortic valves and in cultured aortic valve myofibroblasts, in which their expression was induced by inflammation as well as by cigarette smoke. In conclusion, our findings revealed several novel mechanisms of inflammation (mast cells and mast cell-derived mediators, complement activation), fibrosis (ACE, chymase, cathepsin G, NEP), and elastin fiber degradation (cathepsins) in stenotic aortic valves and highlighted these effectors as possible pathogenic contributors to AS. These results support the notion of AS as an active process with inflammation and extracellular matrix remodeling as its key features and identify possible new targets for medical therapy in AS.

Complement factor C4 and immunoglobulins in recurrent or chronic mucosal infections

The study assessed whether plasma concentrations of complement factors C3, C4, or immunoglobulins, serum classical pathway hemolytyic activity, or polymorphisms in the class I and II HLA genes, isotypes and gene numbers of C4, or allotypes of IgG1 and IgG3 heavy chain genes were associated with severe frequently recurring or chronic mucosal infections. According to strict clinical criteria, 188 consecutive voluntary patients without a known immunodeficiency and 198 control subjects were recruited. Frequencies of low levels in IgG1, IgG2, IgG3 and IgG4 were for the first time tested from adult general population and patients with acute rhinosinusitis. Frequently recurring intraoral herpes simplex type 1 infections, a rare form of the disease, was associated with homozygosity in HLA -A*, -B*, -C*, and -DR* genes. Frequently recurrent genital HSV-2 infections were associated with low levels of IgG1 and IgG3, present in 54% of the recruited patients. This association was partly allotype-dependent. The G3mg,G1ma/ax haplotype, together with low IgG3, was more common in patients than in control subjects who lacked antibodies against herpes simplex viruses. This is the first found immunogenetic deficiency in otherwise healthy adults that predisposes to highly frequent mucosal herpes recurrences. According to previous studies, HSV effectively evades the allotype G1ma/ax of IgG1, whereas G3mg is associated with low IgG3. Certain HLA genes were more common in patients than in control subjects. Having more than one C4A or C4B gene was associated with neuralgias caused by the virus. Low levels of IgA, IgG1, IgG2, IgG3, and IgG4 were common in the general adult population, but even more frequent in patients with chronic sinusitis. Only low IgG1 was more common chronic than in acute rhinosinusitis. Clinically, nasal polyposis and bronchial asthma were associated with complicated disease forms. The best differentiating immunologic parameters were C4A deficiency and the combination of low plasma IgG4 together with low IgG1 or IgG2, performing almost equally. The lack of C4A, IgA, and IgG4, all known to possess anti-inflammatory activity, together with a concurrently impaired immunity caused by low subclass levels, may predispose to chronic disease forms. In severe chronic adult periodontitis, any C4A or C4B deficiency combined was associated with the disease. The new quantitative analysis of C4 genes and the conventional C4 allotyping method complemented each other. Lowered levels of plasma C3 or C4 or both, and serum CH50 were found in herpes and periodontitis patients. In rhinosinusitis, there was a linear trend with the highest levels found in the order: acute > chronic rhinosinusitis > general population > blood donors with no self-reported history of rhinosinusitis. Complement is involved in the defense against the tested mucosal infections. Seemingly immunocompetent patients with chronic or recurrent mucosal infections frequently have subtle weaknesses in different arms of immunity. Their susceptibility to chronic disease forms may be caused by these. Host s subtly impaired immunity often coincides with effective immune evasion from the same arms of immunity by the disease-causing pathogens. The interpretation of low subclass levels, if no additional predisposing immunologic factors are tested, is difficult and of limited value in early diagnosis and treatment.