Determination of the glycosaminoglycan and collagen contents in tissue samples by high-resolution 1H NMR spectroscopy after DCl-induced hydrolysis

The determination of the collagen and glycosaminoglycan (GAG) contents of native and particularly bioengineered tissues is of considerable interest because the collagen-to-GAG ratio determines the water content of the tissue, which is crucial regarding its mechanical properties. (1)H NMR spectroscopy subsequent to the hydrolysis of the sample by aqueous 6 M DCl at 353 K is used to determine the GAG and collagen contents simultaneously. Under these strongly acidic conditions the biopolymers of the extracellular matrix, collagen, and GAG are fragmented into their individual monomers, that is, free amino acids from collagen and monosaccharides from the polymer repeat units of GAGs. The amino acid amount can be easily determined in the presence of an internal standard by (1)H NMR spectroscopy because amino acids proved to be stable under acidic conditions. The carbohydrates are subject to charring in the presence of concentrated DCl, but glucosamine and galactosamine were found to be sufficiently stable for quantification under the chosen conditions.     

Fast and effective: intense pulse light photodynamic inactivation of bacteria

The goal of this study was to investigate the photodynamic toxicity of TMPyP (5, 10, 15, 20-Tetrakis (1-methylpyridinium-4-yl)-porphyrin tetra p-toluenesulfonate) in combination with short pulses (ms) of an intense pulse light source within 10 s against Bacillus atrophaeus, Staphylococcus aureus, Methicillin-resistant S. aureus and Escherichia coli, major pathogens in food industry and in health care, respectively. Bacteria were incubated with a photoactive dye (TMPyP) that is subsequently irradiated with visible light flashes of 100 ms to induce oxidative damage immediately by generation of reactive oxygen species like singlet oxygen. A photodynamic killing efficacy of up to 6 log(10) (>99.9999%) was achieved within a total treatment time of 10 s using a concentration range of 1-100 μmol TMPyP and multiple light flashes of 100 ms (from 20 J cm(-2) up to 80 J cm(-2)). Both incubation of bacteria with TMPyP alone or application of light flashes only did not have any negative effect on bacteria survival. Here we could demonstrate for the first time that the combination of TMPyP as the respective photosensitizer and a light flash of 100 ms of an intense pulsed light source is enough to generate sufficient amounts of reactive oxygen species to kill these pathogens within a few seconds. Increasing antibiotic resistance requires fast and efficient new approaches to kill bacteria, therefore the photodynamic process seems to be a promising tool for disinfection of horizontal surfaces in industry and clinical purposes where savings in time is a critical point to achieve efficient inactivation of microorganisms.     

Production and Regulation of Cuticle-Degrading Proteases from <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in the Presence of <Emphasis Type="Italic">Rhammatocerus schistocercoides</Emphasis> Cuticle

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.     

Structure and active site residues of PglD, an N-acetyltransferase from the bacillosamine synthetic pathway required for N-glycan synthesis in Campylobacter jejuni

Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2,4-diacetamido-2,4,6-trideoxy-d-Glc (QuiNAc4NAc or N,N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 A resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal alpha/beta-domain and a C-terminal left-handed beta-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the beta-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the beta-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His125'.     

Hydroxyurea‐induced partial mushroom body ablation in the honeybee Apis mellifera: Volumetric analysis and quantitative protein determination

Hydroxyurea (HU) treatment of first instar honeybee larvae was previously shown to cause mushroom body (MB) ablations. Predominantly, either one or both median MB subunits were ablated. This prompted us to analyze the effects of asymmetrical or symmetrical HU‐induced MB ablation on both the morphology of the brain and on the level of three proteins (synapsin, PKA RII, and PKC), which are considered to play a role in synaptic plasticity, learning, and memory. In brains with one median MB subunit missing the volume of the overall MB calyx neuropil in the lesioned side was diminished by 35%. This strong reduction occurred although the remaining lateral MB calyx of the lesioned brain side was found to be significantly larger than that of the intact side. Accordingly, in brains with both median MB subunits missing the size of the remaining lateral calyces increased. The various types of MB ablation differentially affected the amounts of synapsin, PKA RII, and PKC expressed in the central brain. In animals with bilateral and thus symmetrical MB ablation (both median calyces ablated) the protein amount was found to be similar to that in control animals. However, unilateral MB ablation causes an increase in the amounts of the tested proteins in the intact brain side, while the levels in the ablated side were the same as in control animals. These findings not only show that HU‐induced ablation of MB subunits is accompanied by volume changes and by changes in protein expression, but also suggest that these processes are highly regulated between the brain sides. The latter is of general importance in understanding the potential contribution of the MB subunits to learning and memory and their interaction between the brain sides. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 31–44, 2002