The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.     

Normal vision can compensate for the loss of the circadian clock

Circadian clocks are thought to be essential for timing the daily activity of animals, and consequently increase fitness. This view was recently challenged for clock-less fruit flies and mice that exhibited astonishingly normal activity rhythms under outdoor conditions. Compensatory mechanisms appear to enable even clock mutants to live a normal life in nature. Here, we show that gradual daily increases/decreases of light in the laboratory suffice to provoke normally timed sharp morning (M) and evening (E) activity peaks in clock-less flies. We also show that the compound eyes, but not Cryptochrome (CRY), mediate the precise timing of M and E peaks under natural-like conditions, as CRY-less flies do and eyeless flies do not show these sharp peaks independently of a functional clock. Nevertheless, the circadian clock appears critical for anticipating dusk, as well as for inhibiting sharp activity peaks during midnight. Clock-less flies only increase E activity after dusk and not before the beginning of dusk, and respond strongly to twilight exposure in the middle of the night. Furthermore, the circadian clock responds to natural-like light cycles, by slightly broadening Timeless (TIM) abundance in the clock neurons, and this effect is mediated by CRY.     

The immunohistochemical demonstration of thyroglobulin in human thyroid tumour xenografts using a monoclonal antibody

A monoclonal antibody (RBU/01) was raised against human thyroglobulin and its suitability for the immunohistochemical staining of thyroglobulin was determined on fixed, wax-embedded tissue, using the peroxidase anti-peroxidase (PAP) method. The antibody was then used to demonstrate the expression of human thyroglobulin in sections of a human follicular carcinoma of the thyroid which had been grown in immunodeficient mice. It is concluded that the immunohistochemical evaluation of the xenografts with the antibody provides useful information on this xenograft system as a potential model for thyroid carcinoma.     

Temporal study of the staphylococci and micrococci of normal infant skin

Staphylococcus and Micrococcus populations were collected from the healthy skin of 10 infant subjects. Infants were sampled from 1 day to 32 weeks of age. Species were characterized by approximately 30 different morphological, physiological, and biochemical characters. Staphylococci were the predominant inhabitants of normal skin, whereas micrococci were found only occasionally in this environment. Staphylococcus epidermidid, S. haemolyticus, and S. hominis were the predominant and persistent staphylococci. These species constituted a high percentage of the total aerobic bacterial flora of infant skin. Micrococcus luteus and M. kristinae were the prevalent micrococci found on infant skin. Only limited correlation between Staphyloccus and Micrococcus populations and infant age or body area sampled was indicated by this study.     

Development and characterization of a human cell line from an ovarian mixed mullerian tumor (carcinosarcoma)

Summary A cell line derived from a human ovarian carcinosarcoma was established in tissue culture and in nude mice. Two sublines, LDF and HDF, separated by discontinuous density centrifugation were also established from the parent line JoN. The cloning efficiency of the JoN line was 21%. Morphologic features of adenocarcinoma cells characteristic of the parent JoN cells were retained in the sublines and clones; all lines showed the same karyotype and DNA content (pseudodiploid and pseudotetraploid). Keratin, as demonstrated immunohistochemically, was strongly expressed in the parent line JoN and the xenograft tumor, but not at all in the LDF sublines and only moderately in the HDF sublines. Vimentin, however, was expressed in neither the parent line JoN nor the xenograft tumor, but was present in both sublines. Transglutaminase and plasminogen activator activity was high in the parent line JoN. Neither, sublines nor clones showed the same high enzyme activity as the parent line. It is concluded that this human tumor line JoN is comprised of epithelial cells, capable of multidirectional differentiation.     

Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.