The Fascinating Coat Surrounding Mycobacteria

The mycobacterial cell envelope is fascinating in several ways. First, its composition is unique by the exceptional lipid content, which consists of very long-chain (up to C90) fatty acids, the so-called mycolic acids, and a variety of exotic compounds. Second, these lipids are atypically organized into a Gram-negative-like outer membrane (mycomembrane) in these Gram-positive bacteria, as recently revealed by CEMOVIS, and this mycomembrane also contains pore-forming proteins. Third, the mycolic acids esterified a holistic heteropolysaccharide (arabinogalacan), which in turn is linked to the peptidoglycan to form the cell wall skeleton (CWS). In slow-growing pathogenic mycobacterial species, this giant structure is surrounded by a capsular layer composed mainly of polysaccharides, primarily a glycogen-like glucan. The CWS is separated from the plasma membrane by a periplasmic space. A challenging research avenue for the next decade comprises the identification of the components of the uptake and secretion machineries and the isolation and biochemical characterization of the mycomembrane.

Molecular architecture of basement membranes

Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.

Electrophoretically mediated microanalysis for characterization of the enantioselective CYP3A4 catalyzed N-demethylation of ketamine

Execution of an enzymatic reaction performed in a capillary with subsequent electrophoretic analysis of the formed products is referred to as electrophoretically mediated microanalysis (EMMA). An EMMA method was developed to investigate the stereoselectivity of the CYP3A4-mediated N-demethylation of ketamine. Ketamine was incubated in a 50 μm id bare fused-silica capillary together with human CYP3A4 Supersomes using a 100 mM phosphate buffer (pH 7.4) at 37°C. A plug containing racemic ketamine and the NADPH regenerating system including all required cofactors for the enzymatic reaction was injected, followed by a plug of the metabolizing enzyme CYP3A4 (500 nM). These two plugs were bracketed by plugs of incubation buffer to ensure proper conditions for the enzymatic reaction. The rest of the capillary was filled with a pH 2.5 running buffer comprising 50 mM Tris, phosphoric acid, and 2% w/v of highly sulfated γ-cyclodextrin. Mixing of reaction plugs was enhanced via application of -10 kV for 10 s. After an incubation of 8 min at 37°C without power application (zero-potential amplification), the capillary was cooled to 25°C within 3 min followed by application of -10 kV for the separation and detection of the formed enantiomers of norketamine. Norketamine formation rates were fitted to the Michaelis-Menten model and the elucidated values for V(max) and K(m) were found to be comparable to those obtained from the off-line assay of a previous study.

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also 'leaking' as revealed by a β-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.

Effects of medetomidine and its active enantiomer dexmedetomidine on N-demethylation of ketamine in canines determined in vitro using enantioselective capillary electrophoresis.

Cytochrome P450 (CYP) enzymes catalyze the metabolism of both, the analgesic and anesthetic drug ketamine and the α2 -adrenergic receptor-agonist medetomidine that is used for sedation and analgesia. As racemic medetomidine or its active enantiomer dexmedetomidine are often coadministered with racemic or S-ketamine in animals and dexmedetomidine together with S- or racemic ketamine in humans, drug-drug interactions are likely to occur and have to be characterized. Enantioselective CE with highly sulfated γ-cyclodextrin as chiral selector was employed for analyzing in vitro (i) the kinetics of the N-demethylation of ketamine mediated by canine CYP3A12 and (ii) interactions occurring with racemic medetomidine and dexmedetomidine during coincubation with ketamine and canine liver microsomes (CLM), canine CYP3A12, human liver microsomes (HLM), and human CYP3A4. For CYP3A12 without an inhibitor, Michaelis-Menten kinetics was determined for the single enantiomers of ketamine and substrate inhibition kinetics for racemic ketamine. Racemic medetomidine and dexmedetomidine showed an inhibition of the N-demethylation reaction in the studied canine enzyme systems. Racemic medetomidine is the stronger inhibitor for CLM, whereas there is no difference for CYP3A12. For CLM and CYP3A12, the inhibition of dexmedetomidine is stronger for the R- compared to the S-enantiomer of ketamine, a stereoselectivity that is not observed for CYP3A4. Induction is observed at a low dexmedetomidine concentration with CYP3A4 but not with CYP3A12, CLM, and HLM. Based on these results, S-ketamine combined with dexmedetomidine should be the best option for canines. The enantioselective CE assay with highly sulfated γ-cyclodextrin as chiral selector is an effective tool for determining kinetic and inhibition parameters of metabolic pathways.