Biomedical subjects

Michel H J Koch

Publications and source records attributed to Michel H J Koch.

At least 19 recordsLinked to original sources

PubMed2006

Structural transformations during gelatinization of starches in limited water: combined wide- and small-angle X-ray scattering study.

Rice flour (18-25% moisture) and potato starch (20% moisture) were heated with continuous recording of the X-ray scattering during gelatinization. Rice flours displayed A-type crystallinity, which gradually decreased during gelatinization. The development of the characteristic 9 nm small-angle X-ray scattering (SAXS) peak during heating at sub-gelatinization temperatures indicated the gradual evolution into a stacked lamellar system. At higher temperatures, the crystalline and lamellar order was progressively lost. For potato starch (B-type crystallinity), no 9 nm SAXS peak was observed at ambient temperatures. Following the development of lamellar structures at sub-gelatinization temperatures, B-type crystallinity and lamellar order was lost during gelatinization. On cooling of partially gelatinized potato starch, A-type crystallinity steadily increased, but no formation of stacked lamellar structures was observed. Results were interpreted in terms of a high-temperature B- to A-type recrystallization, in which the lateral movement of double helices was accompanied by a shift along their helical axis. The latter is responsible for the inherent frustration of the lamellar stacks.

Chemical Phenomena↗

PubMed2006

Gelatinization of starch in excess water: beyond the melting of lamellar crystallites. A combined wide- and small-angle X-ray scattering study.

The gelatinization of waxy rice, regular rice, and potato starch suspensions (66% w/w moisture) was investigated by real-time small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) during heating and by fast ramp differential scanning calorimetry (DSC). The high-angle tail of the SAXS patterns suggested the transition from surface to mass fractal structures in the DSC gelatinization range. Amylose plays a major role in determining the dimensions of the self-similar structures that develop during this process as the characteristic power-law scattering behavior extends to lower scattering angles for regular than for waxy starches. Crystallinity of A-type starches is lost in the temperature region roughly corresponding to the DSC gelatinization range. At the end of the gelatinization endotherm, the B-type potato starch showed residual crystallinity (WAXD), while SAXS-patterns exhibited features of remaining lamellar stacks. Results indicate that the melting of amylopectin crystallites during gelatinization is accompanied by the (exothermic) formation of amorphous networks.

Amylose↗

PubMed2006

Endotoxin-like properties of a rhamnolipid exotoxin from Burkholderia (Pseudomonas) plantarii: immune cell stimulation and biophysical characterization.

Here we report on the purification, structural characterization, and biological activity of a glycolipid, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-alpha(R)-3-hydroxytetradecanoyl-(R)-3-hydroxytetradecanoate (RL-2,2(14)) produced by Burkholderia (Pseudomonas) plantarii. RL-2,2(14) is structurally very similar to a rhamnolipid exotoxin from Pseudomonas aeruginosa and identical to the rhamnolipid of Burkholderia pseudomallei, the causative agent of melioidosis. Interestingly, RL-2,2(14) exhibits strong stimulatory activity on human mononuclear cells to produce tumor necrosis factor alpha, the overproduction of which is known to cause sepsis and the septic shock syndrome. Such a property has not been noted so far for rhamnolipid exotoxins, only for bacterial endotoxins (lipopolysaccharide, LPS). Consequently, we analyzed RL-2,2(14) with respect to its pathophysiological activities as a heat-stable extracellular toxin. Like LPS, the cell-stimulating activity of the rhamnolipid could be inhibited by incubation with polymyxin B. However, immune cell activation by RL-2,2(14) does nor occur via receptors that are involved in LPS (TLR4) or lipopeptide signaling (TLR2). Despite its completely different chemical structure, RL-2,2(14) exhibits a variety of endotoxin-related physicochemical characteristics, such as a cubic-inverted supramolecular structure. These data are in good agreement with our conformational concept of endotoxicity: intercalation of naturally originating virulence factors into the immune cell membrane leads to strong mechanical stress on integral proteins, eventually causing cell activation.

Bacterial Toxins↗

PubMed2005

X-ray scattering of non-crystalline biological systems using synchrotron radiation.

This tutorial review gives an overview of the progress in the study of non-crystalline systems by X-ray scattering and closely related imaging techniques, made possible by advances in synchrotron radiation sources and instrumentation. A brief introduction to the techniques is followed by the presentation of a variety of recent applications to problems in fundamental and applied research in biochemistry and biophysics and food and pharmaceutical technology.

Animals↗

PubMed2005

Saturated phospholipids promote crystallization but slow down polymorphic transitions in triglyceride nanoparticles.

Some matrix materials proposed for the preparation of solid lipid nanoparticles (e.g. trilaurin) are difficult to crystallize after processing by melt-homogenization. In an attempt to overcome this difficulty, the effect of saturated long-chain phospholipids on the crystallization of nanoparticles based on trilaurin, trimyristin, tripalmitin and tristearin was studied. The phospholipids were used as emulsifiers in combination with sodium glycocholate. Saturated phospholipids increased the crystallization temperature of the triglyceride by several degrees compared to soybean phospholipids. The crystallization pattern was more complex in such systems due to solidification of the phospholipid chains prior to triglyceride crystallization. For most triglycerides, egg lecithin also induced crystallization at higher temperatures than natural soybean lecithin. With trilaurin dispersions, the effect of phospholipids can be utilized to induce crystallization at temperatures relevant for larger scale preparation. The polymorphic transitions of the triglycerides were slower in the presence of egg and saturated lecithin leading to a higher stability of the metastable alpha-form. These effects were particularly pronounced in tristearin systems where a predominant fraction of alpha-phase particles could be observed even after long-term cold storage in dispersions containing hydrogenated soybean lecithin or DPPC. The possibility to prepare triglyceride nanoparticles stable in specific modifications offers new opportunities to study effects of polymorphic form on colloidal stability, drug loading and release properties of such dispersions.

Calorimetry, Differential Scanning↗

PubMed2005

Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity.

The physicochemical properties and biological activities of rough mutant lipopolysaccharides Re (LPS Re) as preformed divalent cation (Mg2+, Ca2+, Ba2+) salt form or as natural or triethylamine (Ten+)-salt form under the influence of externally added divalent cations were investigated using complementary methods: Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopic (FT-IR) measurements for the beta <--> alpha gel to liquid crystalline phase behaviour of the acyl chains of LPS, synchrotron radiation X-ray diffraction studies for their aggregate structures, electron density calculations of the LPS bilayer systems, and LPS-induced cytokine (interleukin-6) production in human mononuclear cells. The divalent cation salt forms of LPS exhibit considerable changes in physicochemical parameters such as acyl chain mobility and aggregate structures as compared to the natural or monovalent cation salt forms. Concomitantly, the biological activity was much lower in particular for the Ca2+- and Ba2+-salt forms. This decrease in activity results mainly from the conversion of the unilamellar/cubic aggregate structure of LPS into a multilamellar one. The reduced activity also clearly correlates with the higher order--lower mobility--of the lipid A acyl chains. Both effects can be understood by an impediment of the interactions of LPS with binding proteins such as lipopolysaccharide-binding protein (LBP) and CD14 due to the action of the divalent cations.

Calorimetry, Differential Scanning↗

PubMed2005

Transformation of vesicular into cubic nanoparticles by autoclaving of aqueous monoolein/poloxamer dispersions.

The ultrastructure of aqueous colloidal dispersions of the cubic monoolein/poloxamer 407/water phase, in particular the particle size distribution and presence of an additional vesicular fraction, highly depends on composition and preparation parameters. Therefore, the effect of autoclaving on such dispersions was investigated. Before autoclaving at 121 degrees C, a dispersion of 4.6% monoolein/0.4% poloxamer predominantly consists of cubic particles beside a fraction of non-cubic particles. The small vesicular particles disappear almost completely upon autoclaving whereas larger particles with cubic structure remain in the sample. In contrast, a 4.4% monoolein/0.6% poloxamer dispersion contains predominantly small vesicular particles before heat treatment. After autoclaving, the majority of the particles is larger and of cubic structure and only a few small non-cubic particles remain. The effect can already be observed at short autoclaving times (e.g., 5 min) but a temperature of at least 90 degrees C is required to induce a major change in the ultrastructure. Results from temperature dependent small angle X-ray diffraction investigations indicate that temperatures corresponding to an isotropic phase are required for particle transformation. Heat treatment of monoolein/poloxamer dispersions can thus be used to transform vesicular dispersions into dispersions of cubic phase or to improve the cubic/non-cubic particle ratio in dispersions already containing particles with cubic internal structure.

Colloids↗

PubMed2005

Effects of urea and trimethylamine-N-oxide (TMAO) on the interactions of lysozyme in solution.

The effect of two physiological cosolutes (urea and trimethylamine-N-oxide) and of KCl on the intermolecular interactions in concentrated lysozyme solutions were studied by synchrotron radiation small angle x-ray scattering. The evolution of the structure factors as a function of cosolute and/or salt concentration was modeled using pair potentials following an approach recently described in the literature. It was found that the structure factors for salt and/or cosolute concentration series at a fixed protein concentration can best be described using a variable depth attractive potential and a constant effective charge rather than a constant attractive potential and a variable effective charge as done in previous work.

Animals↗

PubMed2005

High charge-carrier mobility in pi-deficient discotic mesogens: design and structure-property relationship.

Hexaazatrinaphthylene (HATNA) derivatives with six alkylsulfanyl chains of different length (hexyl, octyl, decyl and dodecyl) have been designed to obtain new potential electron-carrier materials. The electron-deficient nature of these compounds has been demonstrated by cyclic voltammetry. Their thermotropic behaviour has been studied by means of differential scanning calorimetry and polarised optical microscopy. The supramolecular organisation of these discotic molecules has been explored by temperature-dependent X-ray diffraction on powders and oriented samples. In addition to various liquid crystalline columnar phases (Col(hd), Col(rd)), an anisotropic plastic crystal phase is demonstrated to exist. The charge-carrier mobilities have been measured with the pulse-radiolysis time-resolved microwave-conductivity technique. They are found to be higher in the crystalline than in the liquid crystalline phases, with maximum values of approximately 0.9 and 0.3 cm(2) V(-1) s(-1), respectively, for the decylsulfanyl derivative. Mobilities strongly depend on the nature of the side chains.

Journal Article↗

PubMed2005

Synthesis and mesomorphic properties of glycosyl dialkyl- and diacyl-glycerols bearing saturated, unsaturated and methyl branched fatty acid and fatty alcohol chains. Part II. Mesomorphic properties.

The biophysical properties of a series of glycosyl dialkyl- and diacyl-glycerols bearing unsaturated or chiral methyl branched chains in the tail, and di- and trisaccharide carbohydrate headgroups are described. Thermotropism was investigated by polarising microscopy, the lyotropism was investigated by small angle X-ray diffraction and by the contact preparation method, and the gel to liquid crystalline phase transition by FT-IR-spectroscopy. The compounds displayed thermotropic Smectic A (SmA), cubic and columnar phases, whereas in the lyotropic phase diagram lamellar, hexagonal and cubic phases are found. The introduction of unsaturated or methyl branched chains leads to liquid crystallinity at ambient temperature. The difference between the 1,3-oleyl-glycerol maltoside and the corresponding 1,2-oleoyl-glycerol maltoside is small.

Acylation↗

PubMed2005

Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling.

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.

Amino Acid Sequence↗

PubMed2005

Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide.

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide-LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the 'endotoxic principle' of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides.

Alkylation↗

PubMed2005

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity.

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.

Antibodies, Monoclonal↗

PubMed2005

The lipopolysaccharide of Brucella abortus BvrS/BvrR mutants contains lipid A modifications and has higher affinity for bactericidal cationic peptides.

The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic beta(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants.

Acylation↗

PubMed2004

Temperature dependence of the binding of endotoxins to the polycationic peptides polymyxin B and its nonapeptide.

The interaction between endotoxins-free lipid A and various lipopolysaccharide (LPS) chemotypes with different sugar chain lengths-and the polycationic peptides polymyxin B and polymyxin nonapeptide has been investigated by isothermal titration calorimetry between 20 and 50 degrees C. The results show a strong dependence of the titration curves on the phase state of the endotoxins. In the gel phase (<30 degrees C for LPS and <45 degrees C for lipid A), an endothermic reaction is observed, for which the driving force is an entropically driven endotoxin-polymyxin interaction, due to disruption of the ordered water structure and cation assembly in the lipid A backbone and adjacent molecules. In the liquid crystalline phase (>35 degrees C for LPS and >47 degrees C for lipid A) an exothermic reaction takes place, which is mainly due to the strong electrostatic interaction of the polymyxins with the negative charges of the endotoxins, i.e., the entropic change DeltaS is much lower than in the gel phase. For endotoxins with short sugar chains (lipid A, LPS Re, LPS Rc) the stoichiometry of the polymyxin binding corresponds to pure charge neutralization; for the compounds with longer sugar chains (LPS Ra, LPS S-form) this is no longer valid. This can be related to the lower susceptibility of the corresponding bacterial strains to antibiotics.

Anti-Bacterial Agents↗

PubMed2004

Cyclic antimicrobial peptides based on Limulus anti-lipopolysaccharide factor for neutralization of lipopolysaccharide.

Bacterial endotoxin (lipopolysaccharide, LPS) is responsible for the septic shock syndrome. As potential therapeutic agents cyclic cationic antimicrobial peptides of different length, based on the Limulus anti-lipopolysaccharide factor (LALF), were synthesized, and their interaction with LPS was characterized physico-chemically and related to results in biological assays. All peptides inhibited the LPS-induced cytokine production in human mononuclear cells and the Limulus amebocyte lysate in a concentration-dependent way, with the peptide comprising the complete LPS-binding loop of the LALF (cLALF22) being the most effective. The peptides were neither cytotoxic nor hemolytic, except a slight effect of cLALF22. The peptides were able to displace Ca(2+) cations from a LPS monolayer, with cLALF22 being again most effective in accordance with results from isothermal titration calorimetry, in which saturation of binding was observed at an equimolar [cLALF22]:[LPS] ratio, and at a ratio 2-2.5 for the other peptides. For cLALF22, zeta (xi) potential experiments exhibited a complete compensation of the negative charges of LPS, whereas for the other peptides a residual negative potential of -20 to -40mV was found. X-ray diffraction experiments showed that the mixed unilamellar/cubic inverted aggregate structure of the lipid A part of LPS was converted into a multilamellar one. The gel to liquid crystalline phase transition of the acyl chains of LPS was changed upon cLALF22 binding, leading to a clear fluidization, which was not observed or only to a lesser degree for the other peptides. The affinity of the peptides for LPS led to a reduced binding of lipopolysaccharide-binding protein (LBP) to target membranes and hence to an inhibition of cytokine induction in human mononuclear cells.

Antimicrobial Cationic Peptides↗

PubMed2004

Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities.

BACKGROUND: Coxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importance. RESULTS: The lipid A moiety of LPS is tetraacylated and has longer (C-16) acyl chains than most other lipid A from enterobacterial strains. The two ester-linked 3-OH fatty acids found in the latter are lacking. The acyl chains of the C. burnetii endotoxins exhibit a broad melting range between 5 and 25 degrees C for LPS and 10 and 40 degrees C for lipid A. The lipid A moiety has a cubic inverted aggregate structure, and the inclination angle of the D-glucosamine disaccharide backbone plane of the lipid A part with respect to the membrane normal is around 40 degrees. Furthermore, the endotoxins readily intercalate into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP). The endotoxin-induced tumor necrosis factor alpha (TNFalpha) production in human mononuclear cells is one order of magnitude lower than that found for endotoxins from enterobacterial strains, whereas the same activity as in the latter compounds is found in the clotting reaction of the Limulus amebocyte lysate assay. CONCLUSIONS: Despite a considerably different chemical primary structure of the C. burnetii lipid A in comparison with enterobacterial lipid A, the data can be well understood by applying the previously presented conformational concept of endotoxicity, a conical shape of the lipid A moiety of LPS and a sufficiently high inclination of the sugar backbone plane with respect to the membrane plane. Importantly, the role of the acyl chain fluidity in modulating endotoxicity now becomes more evident.

Chromatography, Gas↗

PubMed2004

Ligand-dependent structural changes in the V(1) ATPase from Manduca sexta.

The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations.

Animals↗