Identification of specific miRNAs targeting proteins of the apical junctional complex that simulate the probiotic effect of E. coli Nissle 1917 on T84 epithelial cells

In the intestine, dysregulation of miRNA is associated with inflammation, disruption of the gastrointestinal barrier, and the onset of gastrointestinal disorders. This study identifies miRNAs involved in the maintenance of intercellular junctions and barrier integrity. For the functional identification of barrier affecting miRNAs, we took advantage of the barrier-enforcing effects of the probiotic bacterium Escherichia coli Nissle 1917 (EcN) which can be monitored by enhanced transepithelial resistance (TER). miRNA-profiling of T84 monolayers prior and after co-incubation with EcN revealed for the first time differentially regulated miRNAs (miR-203, miR-483-3p, miR-595) targeting tight junction (TJ) proteins. Using real-time PCR, Western blotting and specific miRNA mimics, we showed that these miRNAs are involved in the regulation of barrier function by modulating the expression of regulatory and structural components of tight junctional complexes. Furthermore, specific inhibitors directed at these miRNA abrogated the disturbance of tight junctions induced by enteropathogenic E. coli (EPEC). The half-maximal inhibitory concentration (IC(50)) was determined to 340 nM by monitoring inhibitor kinetics. In summary, we conclude that specific miRNAs effect regulatory as well as structural proteins of the junctional complex which in turn are involved in the barrier enhancing effect of EcN. Hence, we suggest that the application of miRNAs might be refined and further developed as a novel supportive strategy for the treatment of gastrointestinal disorders.     

Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain.

The rat and human recombinant soluble and membrane-bound catechol O-methyltransferase (S- and MB-COMT, respectively) were expressed using mammalian and baculovirus vectors. Low levels of rat and human S-COMT polypeptides were detected by immunoprecipitation in K-562 cell lines transfected with the S-COMT vectors. From K-562 cells transfected with the rat MB-COMT construct, both S- and MB-COMT recombinant proteins were detected by a rat COMT-specific anti-serum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S- or MB-COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S- or MB-COMT proteins, respectively. Pulse/chase experiments with [35S]methionine-labelled insect cells infected with the MB-COMT baculovirus showed that the 30-kDa recombinant human MB-COMT polypeptide was not processed into the 25-kDa S-COMT form. Subcellular fractionations of insect cells, followed by immunoblotting with COMT antiserum, showed that recombinant S-COMT was found only in the soluble, cytoplasmic fraction, whereas MB-COMT resided both in soluble and membrane fractions. The recombinant MB-COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma-membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S-COMT (24 kDa) and some of the rat MB-COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB-COMT polypeptide. The rat brain microsomal MB-COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma-membrane and rough-endoplasmic-reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed.     

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H₂O₂) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H₂O₂ formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H₂O₂ formation during acidic conditions (pH: 3.5-5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H₂O₂ concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H₂O₂ accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H₂O₂ formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H₂O₂. Ascorbic acid/Cu(II)-induced H₂O₂ formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.     

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.     

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants.     

Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer

The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.     

Attitudes to and knowledge about oral health care among nursing home personnel--an area in need of improvement

doi: 10.1111/j.1741‐2358.2011.00562.x
Attitudes to and knowledge about oral health care among nursing home personnel – an area in need of improvement Background: In 1999, a dental reform became law in Sweden that regulated both dental care to dependent individuals and training in oral health care for nursing home personnel. Substantial resources have been channelled into these efforts, but the outcome of these efforts has not been evaluated. The aim of this study was to explore attitudes to and knowledge about oral health care among nursing home personnel more than 5 years after the law was adopted, that being 2005. Methods: A total of 454 individuals employed at nursing homes answered a questionnaire of 16 multiple‐choice items concerning attitudes to and knowledge about oral health care. Results: Eighty‐nine per cent considered oral health care to be an important part of good nursing. The answers indicated problems, however, when it came to its implementation and knowledge, and 35% stated that they had had no formal education in oral health care. Conclusions: Despite generally positive oral health care attitudes, it is important that oral health care education is available to and made of interest for all nursing home personnel, especially in light of the increase in number of natural teeth and frequency of crowns and bridges among dependent elderly.