Types Of Glycolipids Research Articles

Nanomapping of CD1d–glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging

CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.

Inhibition of pathogenic bacterial biofilm by biosurfactant produced by Lysinibacillus fusiformis S9

A biosurfactant producing microbe isolated from a river bank was identified as Lysinibacillus fusiformis S9. It was identified with help of biochemical tests and 16S rRNA gene phylogenetic analysis. The biosurfactant S9BS produced was purified and characterized as glycolipid. The biosurfactant showed remarkable inhibition of biofilm formation by pathogenic bacteria like Escherichia coli and Streptococcus mutans. It was interesting to note that at concentration of 40 μg ml(-1) the biosurfactant did not show any bactericidal activity but restricted the biofilm formation completely. L. fusiformis is reported for the first time to produce a glycolipid type of biosurfactant capable of inhibiting biofilm formation by pathogenic bacteria. The biosurfactant inhibited bacterial attachment and biofilm formation equally well on hydrophilic as well as hydrophobic surfaces like glass and catheter tubing. This property is significant in many biomedical applications where the molecule should help in preventing biofouling of surfaces without being toxic to biotic system.

Characterizing the Binding Kinetics of X31 Influenza Virus to Glycolipids using Single-Particle Tracking

The infection process for enveloped viruses begins with the attachment of viruses to the host cell surface, followed by the fusion of the viral membrane with the host membrane. For Influenza, attachment and fusion are mediated by a single protein, hemagglutinin (HA), anchored in the viral membrane. Successful virus attachment to the host cell hinges on several factors such as the chemical affinity of HA to sialic acid (SA) receptors, polyvalent binding, and binding site accessibility. To characterize binding kinetics of influenza X31 (H3N2), total internal reflection microscopy is used to observe dye-labeled viruses interacting with SA receptors embedded in a supported lipid bilayer. An important advantage of the single-particle binding assay is that it can extract the binding and unbinding times of individual viruses, which are needed to determine the binding residence time distribution and binding rate constant (kon). The binding residence time distribution is used to characterize the binding affinity of HA to SA and binding avidity of a virus to several SA receptors. Meanwhile, the binding rate constant is used to quantify the accessibility of HA to SA.In this work, we examine influenza binding to several types of glycolipids. The glycolipids are GM3, GM1, and GD1a, which all have the alpha 2-3 linked sialic acid group but with varying peripheral glycan structures. Results show that the binding rate is the greatest for GD1a, followed by GM3 and then GM1. The binding residence time data, however, shows little difference between the three receptors. We will discuss possible rationales for these observations, while demonstrating the utility of the single particle assay as a convenient tool for studying binding kinetics.

Autoimmune T-Cell Reactivity to Myelin Proteolipids and Glycolipids in Multiple Sclerosis

Central nervous system (CNS) myelin, the likely major target of autoimmune attack in multiple sclerosis (MS), contains a number of unique components that are potential targets of the attack. Two classes of molecules that are greatly enriched in CNS myelin compared to other parts of the body are certain types of proteolipids and glycolipids. Due to the hydrophobic nature of both of these classes of molecules, they present challenges for use in immunological assays and have therefore been somewhat neglected in studies of T-cell reactivity in MS compared to more soluble molecules such as the myelin basic proteins and the extracellular domain of myelin oligodendrocyte glycoprotein. This review firstly looks at the makeup of CNS myelin, with an emphasis on proteolipids and glycolipids. Next, a retrospective of what is known of T-cell reactivity directed against proteolipids and glycolipids in patients with MS is presented, and the implications of the findings are discussed. Finally, this review considers the question of what would be required to prove a definite role for autoreactivity against proteolipids and glycolipids in the pathogenesis of MS.

Divergent Effects of Mycobacterial Cell Wall Glycolipids on Maturation and Function of Human Monocyte-Derived Dendritic Cells

Background Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system.Principal FindingsWe investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect.ConclusionsThese results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.

Production and characterization of biosurfactant produced by a novel Pseudomonas sp. 2B

Biosurfactant-producing bacteria were isolated from terrestrial samples collected in areas contaminated with petroleum compounds. Isolates were screened for biosurfactant production using Cetyl Tri Ammonium Bromide (CTAB)–Methylene blue agar selection medium and the qualitative drop-collapse test. An efficient bacterial strain was selected based on rapid drop collapse activity and highest biosurfactant production. The biochemical characteristics and partial sequenced 16S rRNA gene of isolate, 2B, identified the bacterium as Pseudomonas sp. Five different low cost carbon substrates were evaluated for their effect on biosurfactant production. The maximum biosurfactant synthesis (4.97g/L) occurred at 96h when the cells were grown on modified PPGAS medium containing 1% (v/v) molasses at 30°C and 150rpm. The cell free broth containing the biosurfactant could reduce the surface tension to 30.14mN/m. The surface active compound showed emulsifying activity against a variety of hydrocarbons and achieved a maximum emulsion index of 84% for sunflower oil. Compositional analysis of the biosurfactant reveals that the extracted biosurfactant was a glycolipid type, which was composed of high percentages of lipid (∼65%, w/w) and carbohydrate (∼32%, w/w). Fourier transform infrared (FT-IR) spectrum of extracted biosurfactant indicates the presence of carboxyl, hydroxyl and methoxyl functional groups. The mass spectra (MS) shows that dirhamnolipid (l-rhamnopyranosyl-l-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate, Rha-Rha-C10-C10) was detected in abundance with the predominant congener monorhamnolipid (l-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate, Rha-C10-C10). The crude oil recovery studies using the biosurfactant produced by Pseudomonas sp. 2B suggested its potential application in microbial enhanced oil recovery and bioremediation.

Glycopeptidolipids: Immuno‐modulators in greasy mycobacterial cell envelope

Species of opportunistic mycobacteria are the major causative agent for disseminating pulmonary infections in immuno-compromised individuals. These naturally resistant strains recruit a unique type of glycolipid known as glycopeptidolipids (GPLs), noncovalently attached to the outer surface of their thick lipid rich cell envelope. Species specific GPLs constitute the chemical determinants of most nontuberculous mycobacterial serotypes, and their absence from the cell surface confers altered colony morphology, hydrophobicity, and inability to grow as biofilms. The objective of this review is to present a comprehensive account and highlight the renewed interest on this much neglected group of pleiotropic molecules with respect to their structural diversity and biosynthesis. In addition, the role of GPLs in mycobacterial survival, both intracellular and in the environment is also discussed. It also explores the possibility of identifying new targets for intervening Mycobacterium avium complex-related infections. These antigenic molecules have been considered to play a pivotal role in immune suppression and can also induce various cytokine mediated innate immune responses, the molecular mechanism of which remains obscure.

The Novel Neutrophil Differentiation Marker Phosphatidylglucoside Mediates Neutrophil Apoptosis

A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP(3) kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.

Theoretical insights into the catalytic mechanism of β-hexosaminidase

The enzyme (β-Hex) has a specific role in the degradation of a particular type of glycolipid, the GM2 ganglioside. This specificity is also manifested by its unusual mechanism of substrate-assisted catalysis, which is considered to be an alternative pathway for the breakage of glycosidic bonds. We have studied its catalytic mechanism using a small model of the enzyme: substrate Michaelis complex with DFT and MP2/MP3 methods. The results reflect the intrinsic chemical reactivity of the active site, decoupled from the long-range enzyme electrostatic field. The mechanism supports, and adds further atomic detail, on the earlier mechanistic suggestions based on experimental data. Moreover, we also have compared the geometry and full potential energy surface obtained with nine different exchange–correlation functionals. It was surprising that the B3LYP activation energies were lower than the ones from some of the hybrid meta functionals (known by their excellent performance on kinetics) by as much as 10 kcal/mol and lower than the MP2 energies by more than 12 kcal/mol. It is known that B3LYP underestimates barriers but underestimations of this extent are unusual and surprising. The effect of the theoretical method on the geometry, usually supposed to be less significant, had in some cases a relevant influence in the mechanism. Therefore, a more thoughtful choice should be made when choosing a methodology for geometry optimization.

Sophorolipids production by Candida bombicola using dairy industry wastewater

Wastewater obtained from a local dairy industry was simultaneously utilized and treated for the production of sophorolipids (SLs), a glycolipids type of biosurfactant, by the yeast Candida bombicola. The dairy wastewater, after initial characterization, was utilized as media with or without adding extra carbon and nitrogen source in the study. A maximum yield of 62 g/l of SLs was found when the yeast was grown on the dairy wastewater supplemented with glucose (10% w/v), yeast extract (0.2% w/v), and soybean oil (10% v/v) in batch shake flasks. Chemical oxygen demand (COD) analysis of the wastewater during the yeast fermentation revealed more than 80% COD removal efficiency in all the experiments. High yield of SLs together with high COD removal efficiency of the yeast in the present study revealed good potential of the system in pretreating such wastewaters and for the production of valuable bioproducts. In addition, the critical micelle concentration and minimum surface tension against water using the produced SLs were found to be 30 mg/l and 33.6 mN/m, respectively. Further, its stability over a wide range of pH (2–10), temperature (up to 120 min of boiling) and salt concentrations (20% NaCl) confirmed the potential of the produced SLs in environmental applications.

Growth inhibition of Microcystis aeruginosa by a Glycolipid-type compound from Bacillus subtilis C1

We attempted to identify the compound responsible for the growth inhibition of Microcystis aeruginosa occurring when a culture broth of Bacillus subtilis C1 was added to the medium. The active compound was purified from B. subtilis C1 culture broth by adsorption chromatography and HPLC, and was identified as a type of glycolipid based on 1H NMR and MS analyses. The purified active compound completely inhibited the growth of M. aeruginosa at a concentration of 10 microgram/ml. This is the first report of a glycolipid produced by a Bacillus strain that has potential as an agent for the selective control of bloom-forming M. aeruginosa.

Novel Polyoxyethylene-Containing Glycolipids Are Synthesized inCorynebacterium matruchotiiandMycobacterium smegmatisCultured in the Presence of Tween 80

The addition of polyoxyethylene sorbitan monooleate (Tween 80) to a culture of mycobacteria greatly influences cell permeability and sensitivity to antibiotics but very little is known regarding the underlying mechanism. Here we show that Corynebacterium matruchotii (surrogate of mycobacteria) converts Tween 80 to a structural series of polyoxyethylenic acids which are then used to form novel series-2A and series-2B glycolipids. Minor series-3 glycolipids were also synthesized. The polyoxyethylenic acids replaced corynomycolic acids in the cell wall. Correspondingly the trehalose dicorynomycolate content was reduced. MALDI mass spectrometry, MS-MS, 1H-NMR, and 13C-NMR were used to characterize the series-2 glycolipids. Series-2A glycolipid is trehalose 6-C36:2-corynomycolate-6′-polyoxyethylenate and series-2B glycolipid is trehalose 6-C36:2-corynomycolate-6′-furan ring-containing polyoxyethylenate. Mycobacterium smegmatis grown in the presence of Tween 80 also synthesizes series-2 type glycolipids. The synthesis of these novel glycolipids in corynebacteria and mycobacteria should result in gross changes in the cell wall permeability and drug sensitivity.

Sophorolipids from Candida bombicola using mixed hydrophilic substrates: Production, purification and characterization

Sophorolipids (SLs) are glycolipids type of biosurfactants and are produced by the yeast Candida bombicola. Medium containing mixed hydrophilic substrate (deproteinized whey and glucose), yeast extract and oleic acid was investigated in this study for SLs production from the yeast. The produced SL was also purified and characterized in the study. At an optimum combination of the medium constituents, the yeast C. bombicola produced maximum 23.29 ± 0.54 g/l, 25.54 ± 1.01 g/l and 33.32 ± 0.83 g/l of the biosurfactant when fermentation was carried out in batch shake flasks, in bioreactor without pH control and in bioreactor with pH control, respectively. Produced SL was purified by silica gel column chromatography and was characterized using FTIR, 1H NMR and LC–MS whose results revealed it to be (17-hydroxyoctadecenoic)-1′4″-lactone-6′6″-diacetate SL. Further, its critical micelle concentration and minimum surface tension against water were found to be 27.17 mg/l and 34.18 mN/m, respectively. Results of interfacial tension obtained using the SLs between water and either n-hexane, sunflower oil or olive oil proved its ability to solubilize non-aqueous phase liquids in water. Further, the biosurfactant was found to be stable at wide range of pHs, temperature and salt concentrations. The results of emulsifying activity and stability of the product against the tested organic solvents and oils together with its ability to solubilize fat and oil confirmed the potential of the biosurfactant in environmental applications.

Two-dimensional supramolecular assemblies involving neoglycoplipids: Self-organization and insertion properties into Langmuir monolayers

In nature, interfacial molecular recognition and chirality are of fundamental significance for the construction of biological assemblies. Lipid monolayers at liquid interface can be used as biomimetic models for studying molecular interactions in such assemblies. In this article, we will focus on the use of Langmuir monolayers for studying self-organization and insertion properties of several neoglycolipids. Two types of glycolipids have been considered, one in the context of the analysis of glycoconjugates of biological relevance, and one dealing with the ability of some glycoprobes to insert into a monolayer in relation with their efficiency for serving as membrane imaging systems.

Novel characteristics of sophorolipids, yeast glycolipid biosurfactants, as biodegradable low-foaming surfactants

Sophorolipids (SLs) are a family of glycolipid type biosurfactants, which are largely produced by the non-pathogenic yeast, Candida bombicola. In order to investigate the possibility of SLs for industrial use, here we examined the interfacial activities, cytotoxicity and biodegradability of SLs, and compared these properties with those of two lipopeptide type biosurfactants (surfactin and arthrofactin), sodium laurate (soap, SP) and four kinds of chemically synthesized surfactants including two block-copolymer nonionic surfactants (BPs), polyoxyethylene lauryl ether (AE) and sodium dodecyl sulfate (SDS). It was indicated that SLs had extremely low-foaming properties and high detergency comparable with commercially available low-foaming BPs. These interfacial activities of SLs were maintained under 100 ppm water hardness. Cytotoxicity of SLs on human keratinocytes was the same as surfactin, which has already been commercialized as cosmetic material, but higher than BPs. Moreover, biodegradability of SLs using the OECD Guidelines for Testing of Chemicals (301C, Modified MITI Test) displayed that SLs can be classified as "readily" biodegradable chemicals, which are defined as chemicals that are degraded 60% within 28 days under specified test methods. We observed 61% degradation of SLs on the eighth day of cultivation. Our results indicate that SLs are low-foaming surfactants with high detergency, which also exhibit both low cytotoxicity and readily biodegradable properties.

Construction of Effective Receptor for Recognition of Avian Influenza H5N1 Protein HA1 by Assembly of Monohead Glycolipids on Polydiacetylene Vesicle Surface

Fast and sensitive detection of epidemic virus is of the utmost importance for human being in nowadays. Various biosensors have been designed for this goal based on conjugation event between host cell glycolipids and invading virus. However, multihead glycolipids analogous to native receptors on cell surface are known to be very difficult to mimic because of the complexity of chemical synthesis. Here, we developed a new approach where two types of monohead glycolipids, active sialic acid-beta-glucoside (G1) and inactive lactose-beta-glucoside (G2), are embedded onto the surface of a polydiacetylene (PDA) vesicle to mimic native glycolipids on the cell surface. Vesicles prepared in this manner show good selectivity with a 10 ng/mL detection limit and 5 min response time to Hemagglutinin (HA1), which is more sensitive than any HA1 biosensors ever known. Moreover, in the formation of color-changeable vesicles, a very strong synergistic effect between G1 and G2 has been found, offering a novel strategy to construct effective biosensor receptors, as well as a new way to study the surface combination effect that is potentially important to the immunology study of epidemic disease.

Preferential expression of phosphatidylglucoside along neutrophil differentiation pathway

Phosphatidylglucoside (PtdGlc), a new type of glycolipid, was recently identified. We examined PtdGlc expression in normal blood cells and leukemic cells using an anti-PtdGlc monoclonal antibody, DIM-21. Neutrophils, monocytes, HL-60 cells and a subset of cord blood (CB) CD34+ cells, but not erythroblasts, expressed lipid antigen. PtdGlc was preferentially expressed along the neutrophil differentiation pathway of CB CD34+ cells treated with cytokines and HL-60 cells treated with retinoic acid. PtdGlc expression was not increased in HL-60 cells treated with phorbol ester. CB CD34+ cells contained a population of PtdGlc+ cells, and CB CD34+PtdGlc+ cells produced mainly granulocyte-macrophage colonies and a small number of erythroid colonies. A positive correlation between PtdGlc expression and CD15 expression in leukemic cells from patients with acute myeloblastic leukemia was shown. These results indicate that increasing PtdGlc expression is seen with neutrophil maturation.

Antigen‐specific recognition of bacterial antigens by NKT cells

The objective of these studies is to understand the biochemistry and biological significance of microbial glycolipid antigen recognition by natural killer T (NKT) cells, a natural memory lymphocyte population that expresses an invariant T cell antigen receptor (TCR) alpha chain. We have identified two types of glycolipids presented by CD1d and recognized by the NKT cell TCR. Sphingomonas, which are environmental bacteria, have antigenic glycosphingolipids (GSLs), containing a ceramide lipid moiety. The second type of lipid is a glycosylated diacyl glycerol found in Borrelia burgdorferi, a spirochete that causes Lyme disease. Interestingly, mouse and human NKT cells selectively respond to diacylglycerols with different acyl chains, as mouse CD1d prefers to present compounds with shorter and less saturated fatty acids. Therefore, the recognition of these antigens is highly sensitive to the nature of the lipid, which is buried in the CD1d groove. To establish the significance of this response, we analyzed NKT cell deficient mice and controls infected with B. burgdorferi. The NKT cell deficient mice had increased joint inflammation at five weeks after infection and reduced bacterial clearance. Our data therefore strongly suggest that the NKT cells participate in host defense against bacterial pathogens through the recognition of microbial glycolipids by their invariant TCR.

The Sclerotinia sclerotiorum agglutinin represents a novel family of fungal lectins remotely related to the Clostridium botulinum non-toxin haemagglutinin HA33/A

Previous studies indicated that sclerotes of the phytopathogenic Ascomycete Sclerotinia sclerotiorum contain a lectin that based on its molecular structure, specificity and N-terminal amino acid sequence could not be classified yet into any lectin family. Using a combination of molecular cloning, frontal affinity chromatography and molecular modelling the identity of the S. sclerotiorum agglutinin (SSA) was analyzed. Molecular cloning demonstrated that SSA shares no sequence similarity with any known fungal lectin or protein. The lectin is synthesized as a 153 amino acid polypeptide without signal peptide and undergoes apart from the removal of the N-terminal methionine no further processing. Frontal affinity chromatography revealed that the binding site of SSA primarily accommodates a non-reducing terminal GalNAc with a preference for the alpha- over the beta-anomer. SSA also strongly interacts with both glycolipid type glycans with terminal non-reducing Gal or GalNAc and galactosylated N-glycans. SSA shares a residual sequence similarity with part of the non-toxin haemagglutinin HA33/A from Clostridium botulinum. Molecular modeling using the three-dimensional structure of HA33/A as a template indicated that SSA can fold into a similar beta-trefoil domain. Though these results should be interpreted with care it is tempting to speculate that the Sclerotiniaceae lectins thus appear to be structurally related to the ricin-B superfamily. All evidence suggests that SSA represents a novel family of fungal lectins with a unique sequence and sugar-binding properties. Taking into account that orthologues of SSA are fairly common within the family Sclerotiniaceae but could not be identified in any other fungal species one can reasonably conclude that SSA-type lectins are confined to a small taxonomic group of the Ascomycota.

Synthesis of Functionalized Amphiphilic Glycoconjugates and Glycoclusters

AbstractGlycolipids are involved in many cellular recognition processes, so there is a need for their synthesis as well as for the design and preparation of glycolipid mimetics. In addition to the application of glycolipid mimetics in a glycobiological context, they are also of interest in many other fields of research, due to their amphiphilic characters. Our goal was to elaborate a series of known orthogonally functionalized glycomimetics and cluster glycosides into glycomimetics of a glycolipid type, so monosaccharide building blocks such as 3, glycodendrons such as 19, and branched cluster glycosides of the type 24 were ligated with lipophilic molecules by peptide coupling, this conjugation providing a new class of complex amphiphilic glycoconjugates with high molecular diversity. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)