Catabolism of dl-α-phenylhydracrylic,phenylacetic and 3- and 4-hydroxyphenylacetic acid via homogentisic acid in a Flavobacterium sp.

A degradation pathway for dl--phenylhydracrylic, phenylacetic, 3- and 4-hydroxyphenylacetic acid by a Flavobacterium is presented. Experiments with washed cells and enzyme studies revealed that dl--phenylhydracrylic acid in an initial reaction was oxidatively decarboxylated to phenylacetaldehyde. Whole cells oxidized both stereoisomers of phenylhydracrylic acid at different rates. The product phenylacetaldehyde in turn was oxidized to phenylacetic acid. No hydroxylation of phenylacetic acid was detected in cell extracts, but on the basis of experiments with washed cells it is assumed that phenylacetic acid is mainly metabolized via 3-hydroxyphenylacetic acid. This latter product was subsequently hydroxylated yielding the ring-cleavage substrate homogentisate. 4-Hydroxyphenylacetic acid was also degraded via homogentisate. Ringcleavage of homogentisate gave maleylacetoacetate which was further degraded through a glutathione-dependent pathway. Homoprotocatechuate was not an intermediate in the metabolism of dl-phenylhydracrylic acid, phenylacetic, 3- and 4-hydroxyphenylacetic acid metabolism, but it could be hydroxylated aspecifically to 2,4,5-trihydroxyphenylacetic acid by the action of the 3-hydroxyphenylacetic acid-6-hydroxylase.Abbreviations HPLC high-performance liquid chromatography - PHA phenylhydracrylic acid - PA phenylacetic acid - HPA hyxdroxyphenylacetic acid - PMS phenazine methosulphate - PMA phenylmalonic acid - GSH glutathione     

Ubiquitous late competence genes in Bacillus species indicate the presence of functional DNA uptake machineries

Natural competence for genetic transformation, i.e. the ability to take up DNA and stably integrate it in the genome, has so far only been observed in the bacterial kingdom (both in Gram-negative and Gram-positive species) and may contribute to survival under adverse growth conditions. Bacillus subtilis , the model organism for the Bacillus genus, possesses a well-characterized competence machinery. Phylogenetic analysis of several genome sequences of different Bacillus species reveals the presence of many, but not all genes potentially involved in competence and its regulation. The recent demonstration of functional DNA uptake by B. cereus supports the significance of our genome analyses and shows that the ability for functional DNA uptake might be widespread among Bacilli .     

The effects of modeling simplifications on craniofacial finite element models: the alveoli (tooth sockets) and periodontal ligaments

Several finite element models of a primate cranium were used to investigate the biomechanical effects of the tooth sockets and the material behavior of the periodontal ligament (PDL) on stress and strain patterns associated with feeding. For examining the effect of tooth sockets, the unloaded sockets were modeled as devoid of teeth and PDL, filled with teeth and PDLs, or simply filled with cortical bone. The third premolar on the left side of the cranium was loaded and the PDL was treated as an isotropic, linear elastic material using published values for Young's modulus and Poisson's ratio. The remaining models, along with one of the socket models, were used to determine the effect of the PDL's material behavior on stress and strain distributions under static premolar biting and dynamic tooth loading conditions. Two models (one static and the other dynamic) treated the PDL as cortical bone. The other two models treated it as a ligament with isotropic, linear elastic material properties. Two models treated the PDL as a ligament with hyperelastic properties, and the other two as a ligament with viscoelastic properties. Both behaviors were defined using published stress-strain data obtained from in vitro experiments on porcine ligament specimens. Von Mises stress and strain contour plots indicate that the effects of the sockets and PDL material behavior are local. Results from this study suggest that modeling the sockets and the PDL in finite element analyses of skulls is project dependent and can be ignored if values of stress and strain within the alveolar region are not required.     

Quantification of epithelial cell differentiation in mammary glands and carcinomas from DMBA- and MNU-exposed rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.     

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca^2 + binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca^2 + is essential for stability as well as correct folding of the whole protein. In the absence of Ca^2 +, pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca^2 + addition, indicating that these are not dead-end species and that Ca^2 + drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6–7 M urea, relates to Ca^2 + dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca^2 + induced folding is not restricted to the RTX domain. Taken together, Ca^2 + binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation.     

Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.