Homologous metabolic and gene activating routes for vitamins E and K

Vitamins E and K share structurally related side chains and are degraded to similar final products. For vitamin E the mechanism has been elucidated as initial ω-hydroxylation and subsequent β-oxidation. For vitamin K the same mechanism can be suggested analogously. ω-Hydroxylation of vitamin E is catalyzed by cytochrome P450 enzymes, which often are induced by their substrates themselves via the activation of the nuclear receptor PXR. Vitamin E is able to induce CYP3A-forms and to activate a PXR-driven reporter gene. It is shown here that K-type vitamins are also able to activate PXR. A ranking showed that compounds with an unsaturated side chain were most effective, as are tocotrienols and menaquinone-4 (vitamin K₂), which activated the reporter gene 8–10-fold. Vitamers with a saturated side chain, like tocopherols and phylloquinone were less active (2–5-fold activation). From the fact that CYPs commonly responsible for the elimination of xenobiotics are involved in the metabolism of fat-soluble vitamins and the ability of the vitamins to activate PXR it can be concluded that supranutritional amounts of these vitamins might be considered as foreign.     

Resorbable plate osteosynthesis of sagittal split osteotomies with major bone movement

This study evaluates resorbable miniplate osteosyntheses in sagittal split osteotomies with major bone repositioning. Two resorbable 2.0-mm miniplate systems, MacroSorb (Macropore, San Diego, Calif.) and PolyMax (Synthes, Oberdorf, Switzerland), were compared consecutively. Amorphous 70:30 poly-L/DL-lactide copolymer plates sustain continuous hydrolysis through water penetration into the implant body during the first 6 months in situ. This breaks copolymer chains into smaller particles, which later become degraded through phagocytotic cells. Eighteen patients, 10 women and eight men, 16 to 57 years old (average, 27 years) were examined. They had severe dysgnathia caused by congenital craniofacial malformations, systemic disorders, trauma, amelogenesis imperfecta, oligodontia, and other conditions, and they needed five 8- to 10-mm and 13 major 10- to 12-mm repositions. Twelve sagittal split osteotomies were fixed with 12 MacroSorb plates in six patients, and 24 osteotomies were filled with 32 PolyMax plates in 12 patients. Ten mandibular plate, screw, hard-tissue, and soft-tissue specimens were taken at 3, 6, 9, or 12 months postoperatively in secondary operations (e.g., dental implant placement).Follow-up ranged from 4 to 19 months; all osteosyntheses reossified. Four patients showed proximal fragments rotated up to 5 mm sagittally anteriorly and nonaligned burr holes on the postoperative radiogram, suggesting plate fractures or screw pullout. When plate fracture was noted, guided occlusion was maintained 4 weeks after surgery. Occlusal, radiologic, and skeletal results remained stable. After starting fixation with two plates on each side, no more plate fractures were seen. In three other patients, minor skeletal relapses up to 3 mm horizontally resulted. Local histologic inspection of specimens showed thorough osseous union. Screw remnants embedded in bone made screw pullout unlikely; rather, screw-head or plate fractures were found as multiple degraded particles. Microscopy showed a chronic foreign body reaction. Two patients (11 percent) developed a sterile fistula 3 and 4 months after surgery, draining implant debris. Here, the biopsies showed a granulocytic infiltrate that subsided clinically after excisional biopsy. The assignment of MacroSorb plates followed by PolyMax plates was done in an otherwise unchanged treatment protocol. Comparison of the number of patients in each group with stable osteosyntheses and regular healing showed no significant differences by Fisher's exact test (p = 0.1516); therefore, the authors focused on the combined results for both treatments.The current osteosynthesis systems showed sufficient stability for mandibular fixation after sagittal split osteotomy and repositioning more than 10 mm distant when two plates were applied to each side; however, 27 percent of patients had complications, including relapses. Disadvantages were the cost, breakability, diameter, and need to place the screws vertically to the plate, necessitating a bent instrument or transbuccal incisions.     

Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3’ portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.     

Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux

Background  

Human and animal health is constantly under threat by emerging pathogens that have recently acquired genetic determinants that enhance their survival, transmissibility and virulence. We describe the construction and development of an Active Surveillance of Pathogens (ASP) oligonucleotide microarray, designed to 'actively survey' the genome of a given bacterial pathogen for virulence-associated genes.     

Structural landscape of isolated agonist-binding domains from single AMPA receptors

AMPA receptors mediate fast excitatory neurotransmission by converting chemical signals into electrical signals, and thus it is important to understand the relationship between their chemical biology and their function. We used single-molecule fluorescence resonance energy transfer to examine the conformations explored by the agonist-binding domain of the AMPA receptor for wild-type and T686S mutant proteins. Each form of the agonist binding domain showed a dynamic, multistate sequential equilibrium, which could be identified only using wavelet shrinkage, a signal processing technique that removes experimental shot noise. These results illustrate that the extent of activation depends not on a rigid closed cleft but instead on the probability that a given subunit will occupy a closed-cleft conformation, which in turn is determined not only by the lowest energy state but also by the range of states that the protein explores.     

The aggregation of mutant p53 produces prion-like properties in cancer

The tumor suppressor protein p53 loses its function in more than 50% of human malignant tumors. Recent studies have suggested that mutant p53 can form aggregates that are related to loss-of-function effects, negative dominance and gain-of-function effects and cancers with a worsened prognosis. In recent years, several degenerative diseases have been shown to have prion-like properties similar to mammalian prion proteins (PrPs). However, whereas prion diseases are rare, the incidence of these neurodegenerative pathologies is high. Malignant tumors involving mutated forms of the tumor suppressor p53 protein seem to have similar substrata. The aggregation of the entire p53 protein and three functional domains of p53 into amyloid oligomers and fibrils has been demonstrated. Amyloid aggregates of mutant p53 have been detected in breast cancer and malignant skin tumors. Most p53 mutations related to cancer development are found in the DNA-binding domain (p53C), which has been experimentally shown to form amyloid oligomers and fibrils. Several computation programs have corroborated the predicted propensity of p53C to form aggregates, and some of these programs suggest that p53C is more likely to form aggregates than the globular domain of PrP. Overall, studies imply that mutant p53 exerts a dominant-negative regulatory effect on wild-type (WT) p53 and exerts gain-of-function effects when co-aggregating with other proteins such as p63, p73 and acetyltransferase p300. We review here the prion-like behavior of oncogenic p53 mutants that provides an explanation for their dominant-negative and gain-of-function properties and for the high metastatic potential of cancers bearing p53 mutations. The inhibition of the aggregation of p53 into oligomeric and fibrillar amyloids appears to be a promising target for therapeutic intervention in malignant tumor diseases.     

PCR-verified microarray analysis and functional in vitro studies indicate a role of α-tocopherol in vesicular transport

Global gene expression profiles of livers from mice, fed diets differing in α-tocopherol content, were compared using DNA microarray technology. Three hundred and eighty nine genes were found to significantly differ in their expression level by a factor of 2 or higher between the high and the low α-tocopherol group. Functional clustering using the EASE software identified 121 genes involved in transport processes. Twenty-one thereof were involved in (synaptic) vesicular trafficking. Up-regulation of syntaxin 1C (Stx1c), vesicle-associated membrane protein 1 (Vamp1), N-ethylmaleimide-sensitive factor (Nsf) and syntaxin binding protein 1 (Stxbp1, Munc18-1) was verified by real time PCR. At a functional level, α-tocopherol increased the secretory response in RBL and PC12 cells. Although here detected in liver, the α-tocopherol-responsive pathways are also relevant to neurotransmission. A role of α-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe α-tocopherol deficiency.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.