Enantioseparation by ligand-exchange using particle-loaded monoliths: capillary-LC versus capillary electrochromatography

Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 microm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of alpha-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with alpha values up to 5.     

Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA

Plasmid DNA for biopharmaceutical applications is mainly produced in E. coli cells. The first and most crucial step for recovering the plasmid is the cell lysis. Governed by the physico-chemical properties of the polynucleotide, alkaline lysis has been the lysis-method of choice. This chemical disintegration technique was initially developed for the lab scale and non-pharmaceutical applications. A continuous, fully automated and closed system combining alkaline lysis, neutralization and clarification in one gentle and generic operation was developed. This system consists of a three units. One unit controls mixing and contact time during the alkaline treatment, another one controls the neutralization and the concurrent formation of flocs and a third one the separation of flocs and pDNA containing lysate. Based on optimization experiments the selected process parameters resulted in yields up to 100% and homogeneities comparable to that obtained by gentle manual lysis. The process does not need enzymes and it is scalable and routinely used for cGMP-production of pharmaceutical grade plasmid DNA from 200 L fermentations.     

Prevention of aneuploidy by S-adenosyl-methionine in human cells treated with sodium arsenite

Alterations in methyl group's metabolism affect availability of S-adenosyl-L-methionine (SAM); these modifications can be originated by enzyme polymorphisms, nutritional deficiencies, and exposure to chemical agents. There are several types of chemicals that interfere with methyl groups, among them is arsenic. It deserves special attention because it modifies a number of cell functions that influence the development of diseases such as cancer. Since part of arsenic's toxicity is influenced by changes on SAM availability, in a previous study we investigated whether exogenous addition of SAM to cells treated with sodium arsenite (NaAsO(2)) has an effect on its genotoxicity. Results demonstrated that SAM reduces the frequency of cells presenting micronuclei (MN) and tubulin-cytoskeleton defects after treatment with NaAsO(2). MN are fragments of the cell nucleus that may contain whole chromosomes or chromosome fragments depending on whether they derive either from the aneugenic or from the clastogenic action of chemicals. Therefore one question generated by these results was whether SAM reduced only the frequency MN resulting from aneugenic damage. To answer this question, in the present work we used an all-centromere DNA probe to distinguish the type of MN reduced by SAM after treatment with NaAsO(2) and vinblastine. In addition, the capacity of SAM to reduce clastogenicity was also evaluated. Results show that SAM decreases the frequency of cells with MN containing whole chromosomes in cultures treated either with NaAsO(2) or with vinblastine; however, induction of double-strand breaks by NaAsO(2) was not prevented by SAM.     

Validation of a voxel-based FE method for prediction of the uniaxial apparent modulus of human trabecular bone using macroscopic mechanical tests and nanoindentation

Assessment of the mechanical properties of trabecular bone is of major biological and clinical importance for the investigation of bone diseases, fractures and their treatments. Finite element (FE) methods are getting increasingly popular for quantifying the elastic and failure properties of trabecular bone. In particular, voxel-based FE methods have been previously used to calculate the effective elastic properties of trabecular microstructures. However, in most studies, bone tissue moduli were assumed or back-calculated to match the apparent elastic moduli from experiments, which often lead to surprisingly low values when compared to nanoindentation results. In this study, voxel-based FE analysis of trabecular bone is combined with physical measures of volume fraction, micro-CT (microCT) reconstructions, uniaxial mechanical tests and specimen-specific nanoindentation tests for proper validation of the method. Cylindrical specimens of cancellous bone were extracted from human femurs and their volume fraction determined with Archimede's method. Uniaxial apparent modulus of the specimens was measured with an improved tension-compression testing protocol that minimizes boundary artefacts. Their microCT reconstructions were segmented to match the measured bone volume fraction and used to create full-size voxel models with 30-45 microm element size. For each specimen, linear isotropic elastic material properties were defined based on specific nanoindentation measurements of its embedded bone tissue. Linear FE analyses were finally performed to simulate the uniaxial mechanical tests. Additional parametric analyses were performed to evaluate the potential errors on the predicted apparent modulus arising from variations in segmentation threshold, tissue modulus, and the use of 125-mm(3) cubic sub-regions. The results demonstrate an excellent correspondence between experimental measures and FE predictions of uniaxial apparent modulus. In conclusion, the adopted voxel-based FE approach is found to be a robust method to predict the linear elastic properties of human cancellous bone, provided segmentation of the microCT reconstructions is carefully calibrated, tissue modulus is known a priori and the entire region of interest is included in the analysis.     

Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen

Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.     

Pathogenicity of entomopathogenic fungi on hibernating pupae of Cameraria ohridella Deschka & Dimic 1986 (Lepidoptera, Gracillariidae). Part 1: Pathogenicity against the naked pupa

Strains from Paecilomyces fumosoroseus, Lecanicillium muscarium, Metarhizium anisopliae and Beauveria bassiana were examined in standardized Biotest to control the horse-chestnut leaf miner (Cameraria ohridella) in her pupal stage in winter. The fungi were pathogenic against the hibernating pupae of Cameraria ohridella at dose of 1.9 x 10(7) conidia/ml. They were aggressive, led to infection, death and mouldiness of naked pupae. Even at low temperature of 5 degrees C and 12 degrees C. L. muscarium strain V24 showed the highest pathogenicity after 4 weeks against this host, close followed by P. fumosoroseus strain P6. M. anisopliae strain 72 and 8. bassiana strain B412 were also pathogen but after a long-time period. Experiments gave information for general susceptibility of naked pupae of C. ohridella under low temperatures against entomopathogenic fungi. In further examinations it has to be tested, whether fungi can infected, when the pupae stay in their natural surroundings, the pupal cell in the leaf.     

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k_cat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.     

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

Background  

The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella.     

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.