Identification of alkane monoxygenase genes inAcinetobacter venetianus VE-C3 and analysis of mutants impaired in diesel fuel degradation

Cells ofAcinetobacter venetianus strain VE-C3 are able to degrade diesel fuel oil by a complex mechanism requiring the formation of cell aggregates and their further adhesion to fuel oil drops. In this work the biodegradation process inA. venetianus was studied by a combination of genetic, molecular and physiological methods. PCR amplification, sequencing and Southern blot analysis ofalkM andrubA genes coding for the alkane hydroxylase and rubredoxin were carried out. Then, 22 Alk^? mutants impaired in diesel fuel degradation were obtained by nitrosoguanidine mutagenesis and characterised by i) growth on alkanes as sole carbon and energy sources, ii) modification of cell electrophoretic properties, and iii) analysis of plasmid content. Data obtained revealed that the genetic determinants for alkane degradation are located on both the chromosome and the two plasmids harboured by VE-C3 strain (pAV1 and pAV2, 11 Kbp and 15 kbp, respectively). This organization of genes coding for alkane monoxygenase complex seems to be similar to the arrangement found in Acinetobacter sp. strains ADP1 and M1, where genes are scattered through the chromosome but, as a novelty, that some genes involved in hydrocarbon degradation are plasmid borne also.     

VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (10(9) cfu/ml) hydrolyzed completely the alpha2-gliadin-derived epitopes 62-75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of alpha2-gliadin-fragment 62-75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3(+) intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.     

Carbonic anhydrase inhibitors. Inhibition of human tumor-associated isozymes IX and cytosolic isozymes I and II with some 1,3,4-oxadiazole-thiols

A series of chiral 1,3,4-oxadiazole-5-thiols incorporating 2-substituted-benzenesulfonamide moieties has been prepared from amino acids, via the ester and carbohydrazide intermediate, followed by cyclization with carbon disulfide. Some of these compounds have been investigated for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the human cytosolic hCA I and II, and the human, transmembrane, tumor-associated isozyme hCA IX. All these compounds showed weak (millimolar) affinity for the three isozymes, except two carbohydrazides and two heterocyclic thiols which selectively inhibited the tumor-associated isozyme with inhibition constants around 10 microM. Such compounds constitute interesting lead molecules for the possible design of CA IX-selective inhibitors.     

Study on the conversion of wool keratin by steam explosion

A wool fiber sample was submitted to chemical-free steam explosion in view of potential exploitation of keratin-based industrial and farm wastes. Fiber keratin was converted into a dark-yellow sludge that was submitted to phase separation by filtration, centrifugation, and precipitation of the soluble materials from the supernatant liquid. The resulting products, when compared with the original wool, showed the extent of disruption of the histology structure, reduction of the molecular weight to water-soluble peptides and free amino acids, and change of the structure of the remainder of the protein associated with breaking of disulfide bonds and decomposition of the high-sulfur-content protein fraction.     

Young microglia restore amyloid plaque clearance of aged microglia

Alzheimer′s disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co‐culturing organotypic brain slices from up to 20‐month‐old, amyloid‐bearing AD mouse model (APPPS1) and young, neonatal wild‐type (WT) mice. Surprisingly, co‐culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.     

Polymorphous granular cells in the lung of the primitive fish,the bichir Polypterus senegalus

This study describes the distribution and the coexpression of specific neurochemical markers in both neuroendocrine‐like cells (NEC‐like) and polymorphous granular cells (PGCs) that populate the mucociliated epithelium of the lung in the air‐breathing fish Polypterus senegalus, using confocal immunohistochemistry. Using confocal immunohistochemistry, we determined the coexpression of specific neurochemical markers. Colocalization studies showed that 5HT is coexpressed with calbindin and nNOS and choline acetyltransferase (ChAT) is coexpressed with nNOS in the PGCs. This study also shows for the first time the simultaneous occurrence of piscidin 1 and 5HT in the PGCs. The function of these cells being equivalent to ones found in fish gill subepithelial parenchyma is still not known. Due to the importance of piscidin 1 in local immune defence, more research is to be useful to understand a possible interaction of PGCs with immune response in the bichir lung. In fact, the capacity of PGCs to produce NO and other neuroactive substances found in immune cells of fish may represent a primitive form of immunoregulation of innate immunity and specifically antimicrobial function as NO induction and respiratory burst activity are correlated with immune response.     

A New Iridoid Dimer and Other Constituents from the Traditional Kurdish Plant Pterocephalus nestorianus Nábělek

Accompanied by other rare compounds, a new iridoid dimer, named kurdnestorianoside ( 1 ), showing an unprecedented secologanol configuration, has been isolated for the first time from the Kurdish medicinal plant Pterocephalus nestorianus, which is used in Kurdistan for treating oral diseases and inflammation. The structure of 1 was established from 1D‐ and 2D‐NMR spectroscopic data. Kaempferol 3‐O‐[3,6‐di‐O‐(E)‐p‐coumaroyl]‐β‐d ‐glucopyranoside ( 7 ) showed a remarkable antiproliferative activity against several human tumor cell lines.     

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.