N-acetylanthranilate amidase from Arthrobacter nitroguajacolicus Rü61a, an alpha/beta-hydrolase-fold protein active towards aryl-acylamides and -esters, and properties of its cysteine-deficient variant

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the alpha/beta-hydrolase-fold superfamily of enzymes; inactivation of (His(6)-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k(cat)/K(m) = 208 mM(-1) s(-1)), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His(6)Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His(6)AmqC22A/C63A markedly differed from those of His(6)Amq. The replacements effected some changes in K(m)s of the enzyme and increased k(cat)s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k(cat) for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in alpha-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.     

Robust computational reconstitution – a new method for the comparative analysis of gene expression in tissues and isolated cell fractions

Background  

Biological tissues consist of various cell types that differentially contribute to physiological and pathophysiological processes. Determining and analyzing cell type-specific gene expression under diverse conditions is therefore a central aim of biomedical research. The present study compares gene expression profiles in whole tissues and isolated cell fractions purified from these tissues in patients with rheumatoid arthritis and osteoarthritis.     

Helices in peptoids of alpha- and beta-peptides

Peptoids of alpha- and beta-peptides (alpha- and beta-peptoids) can be obtained by shifting the amino acid side chains from the backbone carbon atoms of the monomer constituents to the peptide nitrogen atoms. They are, therefore, N-substituted poly-glycines and poly-beta-alanines, respectively. Due to the substituted nitrogen atoms, the ability for hydrogen bond formation between peptide bonds gets lost. It may be very interesting to see whether such non-natural oligomers could be regarded as foldamers, which fold into definite backbone conformers. In this paper, we provide a complete overview on helix formation in alpha- and beta-peptoids on the basis of systematic theoretical conformational analyses employing the methods of ab initio molecular orbital (MO) theory. It can be shown that the alpha- and beta-peptoid structures form helical structures with both trans and cis peptide bonds despite the missing hydrogen bonds. Obviously, the conformational properties of the backbone are more important for folding than the possibility of hydrogen bonding. There are close relationships between the helices of alpha-peptoids and poly-glycine and poly-proline helices of alpha-peptides, whereas the helices of beta-peptoids correspond to the well-known helical structures of beta-peptides as, for instance, the 3(1)-helix of beta-peptides with 14-membered hydrogen-bonded rings. Thus, alpha- and beta-peptoids enrich the field of foldamers and may be used as useful tools in peptide and protein design.     

Psychrobacter faecalis sp. nov., a new species from a bioaerosol originating from pigeon faeces

The taxonomy of strain Iso-46T isolated from a bioaerosol generated by cleaning of a pigeon faeces contaminated room was investigated in a polyphasic approach. The beige pigmented Gram-negative, oxidase-negative organism contained a quinone system with mainly ubiquinone Q-8, and the polar lipid profile was composed of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, beside some hitherto uncharacterized phospholipids. Major polyamines were spermidine and putrescine and also small amounts of cadaverine. The analysis of the fatty acids revealed 3-OH 12:0 and 3-OH 14:0 (within summed feature 3) as hydroxylated fatty acids. These chemotaxonomic characteristics suggest that the strain belongs to the gamma-subclass of the Proteobacteria namely into the genus Psychrobacter. Analysis of the 16S rRNA gene supported the allocation into the genus Psychrobacter, but showing similarities to all described species of this genus lower than 97%. Iso-46T was able to grow on MacConkey agar and other high nutrient containing media within a temperature range of 4 degrees C to 36 degrees C. On the basis of nutritional and further physiological features, a clear differentiation from all other Psychrobacter species was possible. For these reasons it is proposed to create a new species with the name Psychrobacter faecalis sp. nov.     

Novel indeno[1,2-b]indoloquinones as inhibitors of the human protein kinase CK2 with antiproliferative activity towards a broad panel of cancer cell lines

We previously reported indeno[1,2-b]indoles as a novel class of potent inhibitors of the human protein kinase CK2. In the present study we prepared two novel quinoid derivatives, the indeno[1,2-b]indoloquinones 6b and 6c, and demonstrated inhibition of the human CK2 by the compounds. Furthermore, we showed substantial antiproliferative activity of both compounds towards a broad panel of human cancer cell lines in the low micromolar range. Whereas the earlier indeno[1,2-b]indoles have been shown to be selective for CK2, the indeno[1,2-b]indoloquinones 6b and 6c also inhibited the AMPK activated protein kinase ARK5, potentially contributing to the anti-cancer effects of the compounds. In addition, with compound 6b we found a very potent inhibitor of the leukemia-associated receptor tyrosine kinase FLT3, with an IC(50) of 0.18 μM.     

Comparative study of the glycan specificities of cell-bound human tandem-repeat-type galectin-4, -8 and -9

Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galβ1-3GlcNAc or Galβ1-3GalNAc as basic motifs was commonly better than that to canonical Galβ1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay. Fine specificity differences were revealed as follows: (i) gal-8 and -9, but not gal-4, bound to disaccharide Galβ1-3GlcNAc; (ii) increase in binding due to negatively charged substituents was marked only in the case of gal-4 and (iii) gal-4 and -8 bound preferably to histo-blood group A glycans, whereas gal-9 targeted B-type glycans. Experiments with single carbohydrate recognition domains (CRDs) of gal-4 showed that the C-CRD preferably bound to ABH glycans, whereas the N-CRD associated with oligolactosamines. In summary, the comparative analysis disclosed the characteristic profiles of glycan reactivity for the accessible CRD of cell-bound gals. These results indicate the distinct sets of functionality for these three members of the same subgroup of human gals.     

I Dare You to Punish Me—Vendettas in Games of Cooperation

Everybody has heard of neighbours, who have been fighting over some minor topic for years. The fight goes back and forth, giving the neighbours a hard time. These kind of reciprocal punishments are known as vendettas and they are a cross-cultural phenomenon. In evolutionary biology, punishment is seen as a mechanism for maintaining cooperative behaviour. However, this notion of punishment excludes vendettas. Vendettas pose a special kind of evolutionary problem: they incur high costs on individuals, i.e. costs of punishing and costs of being punished, without any benefits. Theoretically speaking, punishment should be rare in dyadic relationships and vendettas would not evolve under natural selection. In contrast, punishment is assumed to be more efficient in group environments which then can pave the way for vendettas. Accordingly, we found that under the experimental conditions of a prisoner’s dilemma game, human participants punished only rarely and vendettas are scarce. In contrast, we found that participants retaliated frequently in the group environment of a public goods game. They even engaged in cost-intense vendettas (i.e. continuous retaliation), especially when the first punishment was unjustified or ambiguous. Here, punishment was mainly targeted at defectors in the beginning, but provocations led to mushrooming of counter-punishments. Despite the counter-punishing behaviour, participants were able to enhance cooperation levels in the public goods game. Few participants even seemed to anticipate the outbreak of costly vendettas and delayed their punishment to the last possible moment. Overall, our results highlight the importance of different social environments while studying punishment as a cooperation-enhancing mechanism.     

Klebsiella oxytoca: opportunistic infections in laboratory rodents

Opportunistic pathogens have become increasingly relevant as the causative agents of clinical disease and pathological lesions in laboratory animals. This study was conducted to evaluate the role of Klebsiella oxytoca as an opportunistic pathogen in laboratory rodents. Therefore, K. oxytoca-induced lesions were studied from 2004 to early 2006 in naturally infected rodent colonies maintained at The Jackson Laboratory (TJL), Bar Harbor, USA, the Animal Research Centre (Tierforschungszentrum, TFZ) of the University of Ulm, Germany and the Central Animal Facility (ZTM) of the Hannover Medical School, Germany. K. oxytoca infections were observed in substrains of C3H/HeJ mice, which carry the Tlr4(Lps-d) allele; in LEW.1AR1-iddm rats, the latter being prone to diabetes mellitus; in immunodeficient NMRI-Foxn1(nu) mice; and in mole voles, Ellobius lutescens. The main lesions observed were severe suppurative otitis media, urogenital tract infections and pneumonia. Bacteriological examination revealed K. oxytoca as monocultures in all cases. Clonality analysis performed on strains isolated at the ZTM and TFZ (serotyping, pulse field gel electrophoresis [PFGE], enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction, sequencing of 16S rRNA and rpoB genes) revealed that the majority of bacteria belonged to two clones, one in each facility, expressing the capsule type K55 (ZTM) or K72 (TFZ). Two strains, one isolated at the ZTM and one at the TFZ, showed different PFGE and ERIC pattern than all other isolates and both expressed capsule type K35. In conclusion, K. oxytoca is an opportunistic pathogen capable of inducing pathological lesions in different rodent species.     

International ethical regulations on placebo-use in clinical trials: a comparative analysis

The ethical aspects of placebo control in clinical trials have been extensively and controversially debated in the last decade. However, a thorough analytical comparison of the different existing international regulations, their terminologies and their ethical principles concerning placebo, is still missing. The central issue in the ongoing controversy is the justification of placebo-use, if proven treatment exists. All present versions of the examined guidelines propose such justifications, but each guideline differs from the others in relevant details. Therefore the conditions justifying placebo-use according to each guideline are the focus of our attention. We will first propose a formalized general principle that defines the ethical acceptability of placebo-use. Then we will analyse three categories of conditions put forward by the different documents: the risk of harm or burden, compelling scientific reasons, and the availability of proven treatment. The analysis shows important normative discrepancies and contradictions between the examined guidelines. Especially striking is the fact that some guidelines allow the participants in clinical trials to be exposed to a risk of serious harm, while others do not. Finally, we try to show how the normative difference of each guideline could influence the decision of researchers or IRBs concerning the ethical acceptability of placebo-use.