Antigenic localities in the tissues of the young adult worm of Paragonimus westermani using immunogold labeling method

In order to observe the antigenic localization in the tissues of the young adult Paragonimus westermani, immunogold labeling method was applied using serum immunoglobulins(IgG) of the dog which infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex (particle size; 12 nm). It was observed by electron microscopy at each tissues of the worm. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were predominantly labeling on the epithelial lamela and lumen of caecum. The above finding showed that antigenic materials in young adult worm tissue were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells.     

Pilot-scale production of l-phenylalanine from d-glucose

Effects of inoculum size and total sugar content on both l-phenylalanine productivity and titre have been investigated using a tyrosine auxotrophic regulatory mutant of Escherichia coli. Fermentations were carried out in a 500 litre pilot fermenter with intermittent feeding of d-glucose plus phosphate. It was found that the productivity was not greatly affected by inoculum size. However, the l-phenylalanine titre was significantly affected by total sugar content. Relatively high productivities of up to 0.35–0.40 g l-phenylalanine l^?1 h^?1 have been achieved at l-phenylalanine titres of 14–15 g l^?1.     

Examining serological diversity of “cowpea” rhizobia by the ELISA technique

To appraise the usefulness of the enzyme-linked immunosorbent assay (ELISA) technique for examining the serological diversity of slow-growing rhizobia, twelve diverse strains from three countries were examined with four antisera. Eleven of the strains were from the cowpea miscellany, and the twelfth was a Rhizobium japonicum strain. Some cowpea strains showed no antigenic relatedness with each other while others were closely related, and some showed a greater affinity with the R. japonicum strain than with other cowpea strains. All of the strains showed antigenic homology to an isolate from a wild Arachis sp., while two strains isolated from adjacent plants of the same cultivar had little homology. These patterns ofrelatedness and diversity clearly demonstrated the utility of the ELISA method, and so it was used to examine 53 strains isolated from cowpeas grown at three West African locations, Maradi (Niger), Ibadan and Onne (Nigeria). Broad ranges of serological diversity were found in the rhizobia at each location, moreover each population had its own general characteristics. Maradi strains were highly reactive with the five antisera used, Onne strains less so, and Ibadan strains even less so. ELISA reactivity correlated with colony morphology but not with nodulation potential.     

Cowpea Rhizobia Producing Dark Nodules: Use in Competition Studies

During a program of screening rhizobia from West Africa, it was found that some strains produced nodules of unusually dark appearance on cowpeas, but not on peanuts, soybeans, pigeon peas, or mung beans. The dark pigmentation was in the bacteroid zone, was not correlated with nodule effectiveness, and was additional to the leghemoglobin pigment. Only rhizobial strains with a nongummy (“dry”) colony morphology produced dark nodules. Visually distinguishable pink and dark nodules formed on the same root when a mixture of pink and dark strains was applied as inoculum. The dark-nodule phenotype was therefore appraised as a marker and found to be useful for studying nodulation competition with strains of the orthodox pink-nodule type. The competitiveness of 10 pink-nodule strains was examined relative to a black-nodule strain, IRc 256; a range of competitiveness was obtained of less competitive than, equally competitive to, or more competitive than IRc 256. Patterns of primary (early) nodulation were generally the same as patterns of secondary (later) nodulation. Mixed infections by dark and pink strains produced piebald nodules, the frequency of occurrence of which was much greater among primary than among secondary nodules.     

Antigenic localities in the tissues of Paragonimus westermani by developmental stages using immunogold labeling method]

In order to observe the antigenic localization in the tissues of Paragonimus westermani of developmental stages, immunogold labeling method was applied using serum of the cats which were infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissues from each developmental stage were embedded in Lowicryl HM 20 medium, stained with infected serum IgG and protein A gold complex (particle size: 12 nm) and observed by electron microscopy. In the young adult worm tissue of 4 weeks after infection with metacercariae, the gold particles were specifically concentrated on the tegumental syncytium and cytoplasm of the tegumental cells as well as the secretory granules in the parenchymal tissue. The antigenic materials in the adult worm tissue were specifically concentrated on the secretory granules in the parenchymal tissue, the cytoplasm between granules in the vitelline gland and the epithelial lamella in the lumen of the caecum.     

Secondary structure of mouse 28S rRNA and general model for the folding of the large rRNA in eukaryotes.

We present a secondary structure model for the entire sequence of mouse 28S rRNA (1) which is based on an extensive comparative analysis of the available eukaryotic sequences, i.e. yeast (2, 3), Physarum polycephalum (4), Xenopus laevis (5) and rat (6). It has been derived with close reference to the models previously proposed for yeast 26S rRNA (2) and for prokaryotic 23S rRNA (7-9). Examination of the recently published eukaryotic sequences confirms that all pro- and eukaryotic large rRNAs share a largely conserved secondary structure core, as already apparent from the previous analysis of yeast 26S rRNA (2). These new comparative data confirm most features of the yeast model (2). They also provide the basis for a few modifications and for new proposals which extend the boundaries of the common structural core (now representing about 85% of E. coli 23S rRNA length) and bring new insights for tracing the structural evolution, in higher eukaryotes, of the domains which have no prokaryotic equivalent and are inserted at specific locations within the common structural core of the large subunit rRNA.     

Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer

Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.     

Effect of inoculation of lactic acid bacteria on proteolytic activity of psychrotrophic Gram-negative bacteria in fresh farmed sea bass (Dicentrarchus labrax) fillets during storage at 4 °C under vacuum-packed conditions

The effect of two strains of lactic acid bacteria (LAB) (Lactococcus lactis and Carnobacterium piscicola) on the proteolytic activity of four strains of Psychrotrophic Gram-negative bacteria [Psy G(?)] (Pseudomonas fluorescens, Aeromonas hydrophila, Pseudomonas putida and Photobacterium damselae) has been determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), in fresh vacuum-packed farmed sea bass (Dicentrarchus labrax) fillets artificially contaminated, during 21 days of chilled storage. The profiles of sarcoplasmic (SP) and myofibrillar (MP) proteins indicated that the major changes were produced with Pseudomonas fluorescens, Aeromonas hydrophila, and Pseudomonas putida starters. The results also showed that LAB strains presented a weak proteolytic activity against MP and SP proteins in muscle of fresh sea bass. In fact, we noted the less pronounced degradation of protein fractions in samples inoculated with LAB combination. Moreover, a significant bacteriostatic effect of LAB strains was demonstrated against all microflora, particularly mesophilic aerobic plate counts (MAPC) and psychrotrophic bacterial counts (PBC), with fillets remaining unspoiled until the end of storage, against values of 7 and 8 log CFU/g, respectively; control fish fillets exceeded the upper acceptability limit.     

Characterisation of a novel Emaravirus identified in mosaic-diseased Eurasian aspen (Populus tremula)

Since Emaraviruses have been discovered in 2007 several new species were detected in a range of host plants. Five genome segments of a novel Emaravirus from mosaic-diseased Eurasian aspen (Populus tremula) have been completely determined. The monocistronic, segmented ssRNA genome of the virus shows a genome organisation typical for Emaraviruses encoding the viral RNA-dependent RNA polymerase (RdRP, 268.2 kDa) on RNA1 (7.1 kb), a glycoprotein precursor (GPP, 73.5 kDa) on RNA2 (2.3 kb), the viral nucleocapsid protein (N, 35.6 kDa) on RNA3 (1.6 kb), and a putative movement protein (MP, 41.0 kDa) on RNA4 (1.6 kb). The fifth identified genome segment (RNA5, 1.3 kb) encodes a protein of unknown function (P28, 28.1 kDa). We discovered that it is distantly related to proteins encoded by Emaraviruses, such as P4 of European mountain ash ringspot-associated virus. All proteins from this group contain a central hydrophobic region with a conserved secondary structure and a hydrophobic amino acid stretch, bordered by two highly conserved positions, thus clearly representing a new group of homologues of Emaraviruses. The virus identified in Eurasian aspen is closely associated with observed leaf symptoms, such as mottle, yellow blotching, variegation and chloroses along veins. All five viral RNAs were regularly detectable by RT-PCR in mosaic-diseased P. tremula in Norway, Finland and Sweden (Fennoscandia). Observed symptoms and testing of mosaic-diseased Eurasian aspen by virus-specific RT-PCR targeting RNA3 and RNA4 confirmed a wide geographic distribution of the virus in Fennoscandia. We could demonstrate that the mosaic-disease is graft-transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft-inoculated seedlings used as rootstocks as well as in the virus-infected scions used for graft-inoculation. Owing to these characteristics, the virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic-associated virus.