Activation of Plasmin in Mastitic Milk

Milk and whey samples from healthy and inflamed udder quarters of 10 Ayrshire cows were analyzed for proteolytic activity using radial caseolysis procedures, a fluorogenic coumaryl peptide substrate, and casein agarose zymography. Free lysosomal enzyme activity (N–acetyl–beta–D–glucosaminidase) was used as the criterion for inflammation. All mastitic milk samples had proteolytic activity, tentatively identified as plasmin (comigration at Mr 83 000 and characteristic fragmentation). The plasmin activities in mastitic milk were on average 2.9 μg/ml (range 0.5–12.5) as measured by radial caseolysis. Milk or whey specimens from healthy quarters were all negative except 1 in which an activity of 0.1 μg/ml was found in both specimens. The caseolytic activities were totally inhibited by 50 KITJ/ml of aprotinin, a serine proteinase inhibitor from bovine lung. No free plasminogen activator (PA) activity was found in any of the samples. Howewer, according to zymographic analyses PA molecules corresponding to urokinase were found in healthy and especially in mastitic specimens. Analysis of plasmin may provide an alternative means of screening for mastitic milk samples.     

Magnetic Fractionation and Proteomic Dissection of Cellular Organelles Occupied by the Late Replication Complexes of Semliki Forest Virus

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection.     

Complex T cell memory repertoires participate in recall responses at extremes of antigenic load

The CD8 T cell memory response to the HLA-A2-restricted influenza epitope M1(58-66) can be an instructive model of immune memory to a nonevolving epitope of a frequently encountered pathogen that undergoes clearance. This memory repertoire can be complex, composed of a large number of clonotypes represented at low copy numbers, while maintaining a focus on the use of VB17 T cell receptors with identified Ag recognition motifs. Such a repertoire structure might provide a panoply of clonotypes whose differential avidity for the epitope would allow responses under varying antigenic loads. This possibility was tested experimentally by characterizing the responding repertoire in vitro while varying influenza Ag concentration over five orders of magnitude. At higher and lower Ag concentrations there was increased cell death, yet a focused but diverse response could still be observed. Thus, one of the characteristics of complex memory repertoires is to provide effector function at extremes of Ag load, a characteristic that is not generally considered in vaccination development but may be important in measuring its efficacy.     

Adult hippocampal cell proliferation is suppressed with estrogen withdrawal after a hormone-simulated pregnancy

Estradiol withdrawal after pregnancy is hypothesized to precipitate depressive symptoms in vulnerable women. A hormone-simulated pregnancy was induced in female rats and the effects of a ‘postpartum’ drop in estradiol on hippocampal cell proliferation were examined. All groups were ovariectomized or given sham surgery prior to treatment. Rats were randomly assigned to ‘postpartum’, ‘postpartum’ + EB (estradiol benzoate), ‘postpartum’ + DPN (diarylpropionitrile; an ERβ agonist), ‘postpartum’ + IMI (imipramine; a tricyclic antidepressant), sham, ovariectomized (OVX), sham + IMI or OVX + IMI groups. All ‘postpartum’ groups received hormone injections (estradiol and progesterone) over 23 days to simulate pregnancy, while IMI groups also received daily imipramine injections. After day 23, ‘postpartum’ rats were withdrawn from the hormone-simulated pregnancy (mimicking the postpartum drop in gonadal hormones), while other ‘postpartum’ treatment groups received daily injections of DPN, EB or IMI. On day 3 ‘postpartum’ all rats were injected with bromodeoxyuridine (BrdU; a DNA synthesis marker) and perfused 24 h later to assess cell proliferation and cell death in the dentate gyrus. ‘Postpartum’ hormone withdrawal decreased hippocampal cell proliferation in the ‘postpartum’ and ‘postpartum’ + EB groups only. Chronic imipramine significantly increased hippocampal cell proliferation in sham + IMI, but not OVX + IMI rats suggesting that imipramine's effects to increase hippocampal cell proliferation in female rats is related to reproductive status. Cell death (pyknotic cells) was decreased only in the ‘postpartum’ group. Together, these results suggest an important, though complex, role for gonadal hormones in the cellular changes accompanying this model of postpartum depression.     

Oxidative d-xylose metabolism of Gluconobacter oxydans

Summary Gluconobacter oxydans subsp. suboxydans ATCC 621 oxidizes d-xylose to xylonic acid very efficiently, although it cannot grow on xylose as sole carbon source. The oxidation of xylose was found to be catalyzed by a membrane-bound xylose dehydrogenase. The xylono--lactone formed in the oxidation reaction is subsequently hydrolyzed to xylonic acid by a -lactonase. The complete oxidation pathway of d-xylose in G. oxydans is evidently located in the periplasmic space.     

Structural analysis of enzyme-resistant isomaltooligosaccharides reveals the elongation of α-(1→3)-linked branches in Weissella confusa dextran

Weissella confusa VTT E-90392 is an efficient producer of a dextran that is mainly composed of α-(1→6)-linked D-glucosyl units and very few α-(1→3) branch linkages. A mixture of the Chaetomium erraticum endodextranase and the Aspergillus niger α-glucosidase was used to hydrolyze W. confusa dextran to glucose and a set of enzyme-resistant isomaltooligosaccharides. Two of the oligosaccharides (tetra- and hexasaccharide) were isolated in pure form and their structures elucidated. The tetrasaccharide had a nonreducing end terminal α-(1→3)-linked glucosyl unit (α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc), whereas the hexasaccharide had an α-(1→3)-linked isomaltosyl side group (α-D-Glcp-(1→6)[α-D-Glcp-(1→6)-α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). A mixture of two isomeric oligosaccharides was also obtained in the pentasaccharide fraction, which were identified as (α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc) and (α-D-Glcp-(1→6)[α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). The structures of the oligosaccharides indicated that W. confusa dextran contains both terminal and elongated α-(1→3)-branches. This is the first report evidencing the presence of elongated branches in W. confusa dextran. The (1)H and (13)C NMR spectroscopic data on the enzyme-resistant isomaltooligosaccharides with α-(1→3)-linked glucosyl and isomaltosyl groups are published here for the first time.     

Localization of cytokinins in somatic and zygotic embryos of Tilia cordata using immunocytochemistry

Immunofluorescent and immunogold labeling was used to study the localization of cytokinins in developing somatic and zygotic embryos of Tilia cordata Miller. Broad-specificity polyclonal antibodies active against dihydrozeatin riboside (DHZR)/zeatin riboside (ZR)-type and isopentenyladenosine (iPR)-type cytokinins were used for immunolabeling. Immunofluorescent microscopy showed that these cytokinins were concentrated in highly cytoplasmic cells showing meristematic character. Cotyledon initials and primary meristems of heart-stage somatic embryos, as well as heart-stage zygotic embryos, were labeled. During elongation of embryos, cytokinin immunoreactive material was concentrated to areas having meristematic character. Root apex, shoot meristem, and cotyledon cells of somatic and zygotic cotyledonary embryos, as well as epidermal and subepidermal cell layers of the hypocotyl, showed the strongest immunoreaction. The nucleoli, especially, had a very strong signal. Results at the ultrastructural level with gold-conjugated protein A supported these conclusions. Gold particles were distributed in the nuclei, especially in the nucleoli and throughout the ground cytoplasm. They were occasionally associated with plastids and mitochondria, but seldom with other organelles.     

RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 × 10⁹ cfu μg^− 1 ml^− 1 with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 × 10⁶ and 1 × 10⁸ cfu μg^− 1 ml^− 1. The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10³ times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10⁶ times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.     

A hidden species becoming visible: biogeography and ecology of Rhynchotalona latens (Cladocera,Anomopoda, Chydoridae)

Hydrobiologia - A long hidden chydorid (Chydoridae, Cladocera) taxon, first found as fossil specimens and recently redefined as Rhynchotalona latens (Sarmaja-Korjonen et al., Hydrobiologia 436:...     

New biocatalytic route for the production of enantioenriched β-alanine derivatives starting from 5- and 6-monosubstituted dihydrouracils

Taking advantage of the catalytic promiscuity of pyrimidine-catabolism enzymes (dihydropyrimidinase (E.C. 3.5.2.2), N-carbamoyl-β-alanine amidohydrolase (E.C. 3.5.1.6)), the production of different β-alanine derivatives starting from 5- and 6-monosubstituted dihydrouracils has been evaluated using a mimesis approach. In this work, the S-enantioselective character of dihydropyrimidinase from Sinorizhobium meliloti toward 6-monosubstituted dihydrouracil derivatives has been shown. An inverted R-/S-enantioselectivity of N-carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens toward two different N-carbamoyl-β-amino acids has been proved. Our results have shown for the first time that this mimetic tandem constitutes an interesting biotechnological tool for the preparation of different β-alanine derivatives in an environmentally friendly way, allowing the production of enantioenriched (R)-α-phenyl-β-alanine (e.e. > 95%) and (R)-α-methyl-β-alanine (e.e. > 90%).