Translational capacity of sheep oocytes microinjected with messenger RNA

Sheep oocytes were microinjected with tobacco mosaic virus RNA (TMV-RNA) and isotopically labelled with L-[35S]methionine. Total incorporation of labelled methionine was similar in TMV-RNA-injected and in carrier-injected control oocytes, whether injections were performed during the period of high protein synthesis at maturation or during the period of reduced synthesis at a time equivalent to the mid-cleavage transition (48 h after germinal vesicle breakdown). Varying the amount of TMV-RNA injected from 2.5 to 10 pg had little effect on the overall level of amino acid incorporation. Furthermore TMV-RNA appeared to be very stable in oocytes and eggs; the proportion of total polypeptide synthesis directed by TMV-RNA did not diminish during the first 48 h after injection. Synthesis of most endogenous proteins was uniformly reduced to compensate for the synthesis of TMV-polypeptides. Our results suggest, therefore, that the translational capacity of sheep oocytes is fully saturated during maturation.     

Production of DON-related trichothecenes byFusarium graminearum Schw from Krasnodarski krai of the USSR

34Fusarium graminearum Schw isolates produced 4-deoxynivalenol to form significant amounts of 4, 7 — dideoxynivalenol and lesser amounts of 4 — deoxynivalenol monoacetates on grain substratesin vitro. This is the first report on the capability a large group of naturally occurring isolates to produce 4,7-dideoxynivalenol. The average levels of 4,7-dideoxynivalenol on rice, corn, barley, and wheat as a substrate were respectively 26.8, 14.0, 12.8, and 10.5% of the level of 4-deoxynivalenol. 4, 7 — dideoxynivalenol was present in all examined naturally contaminated wheat kernel samples at levels of 1.7 to 7.9% of the level of 4-deoxynivalenol. These findings suggest that more attention should be given to the occurrence of 4,7-dideoxynivalenol in cereals.     

In vitro pluripotency of epiblasts derived from bovine blastocysts

Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

Synthesis of intraspecific somatic hybrids of Solanum tuberosum: assessments of morphological, biochemical and nematode (Globodera pallida) resistance characteristics

In an attempt to produce novel agronomic traits, a number ofintraspecific somatic hybrid plants have been produced followingleaf mesophyll protoplast fusion between S. tuberosum dihaploidclones PDH 40 (possessing good tuber shape and yield) and PDH417 (possessing resistance to potato cyst nematode, G. pallida).PDH 417 protoplast-derived calli failed to regenerate plantsusing the described culture conditions preventing this parentaltype amongst the mass of regenerated fusion products. Initially,somatic hybrid plants were selected based on differential pigmentationin tuber sprouts and where possible on petal colour. Differentialmobility of patatin bands in electrophoresed tuber extractsfurther confinned hybridity. The intraspecific somatic hybridsalso showed different levels of resistance to G. pallida pathotypesPa2 and Pa3 in the somatic hybrid plants examined. Key words: Somatic hybridization, dihaploids, patatin, nematode resistance, Solanum tuberosum, potato     

Production and modifications of extracellular structures during development of chytridiomycetes

Summary In development of the primitive fungi, chytridiomycetes, unwalled zoospores bearing single, posterior flagella are transformed into walled, round-cells which elaborate the thallus. Production, structural modification, or release of extracellular material are involved with each transition of developmental stage. This article reviews the variety and developmental changes of extracellular materials found at the cell surface of chytridiomycetes. A cell coat, produced from Golgi-derived vesicles during zoosporogenesis, is visible around free swimming zoospores of some chytridiomycetes. How the zoospore surface receives and transduces signals is not widely explored, but it is known that fenestrated cisternae and simple cisternae, which are integrated into the microbody-lipid globule complex, are spatially and structurally associated with the plasma membrane and flagellar apparatus. This spatial association, as well as the cytochemical localization of calcium in fenestrated cisternae, suggest a mechanism for signal transduction and for regulation of zoospore motility. Zoospores become encased in a new layer of extracellular material as the zoospore encysts. Among some chytrids the source of this material is preexisting vesicles which fuse with the plasma membrane. Among other zoospores, a readily identifiable population of encystment vesicles is not apparent, demonstrating that there is no single pattern or mechanism for zoospore encystment in chytridiomycetes. Encysted zoospores developing into thalli, typically produce cell walls with a microfibrillar substructure. Ultrastructural analysis of walls reveals distinctive architecture and remarkable sculpturing which have been used in systematics of some members of chytridiomycetes. Nothing is known as to underlying controls of cytoskeletal elements and plasma membrane enzyme complexes in wall biogenesis. Many changes in cell surface structures accompany thallus maturation. Septa, many traversed with plasmodesmata, are produced in most chytrid thallus types. As sporangia and resting spores prepare for the production and release of zoospores, additional extracellular layers of material are frequently produced. Polarized deposits of extracellular material become discharge plugs, discharge vesicles, or endoopercula. Interstitial material is also released into cleavage furrows. Circumscissile or localized digestion of walls produce operculate or inoperculate exit ports for zoospore release. Cryofixation preserves more extensive extracellular material than does conventional chemical fixation, and broader application of cryofixation may radically alter our current view of cell surface structure. Thus chytridiomycetes exhibit a range in patterns for the occurrence and subsequent modifications of extracellular materials, even for members within the same order. The most universally recognized role for these extracellular materials is protection. Although there is a reasonable view of the types of extracellular material involved in chytridiomycete development, we have only limited understandings of their biogenesis or roles in regulation and communication, areas awaiting more investigations.Abbreviations DIC Nomarski-differential contrast optics - TEM transmission electron microscopy     

Seed storage protein variation in Arachis species.

Seventy-two accessions, representing 22 species from sections Arachis, Erectoides, Extranervosae, and Triseminalae of the genus Arachis, were screened for seed storage protein polymorphism. Variation was detected between sections, between genome types, between species, and in some cases between different accessions of the same species or different seeds of the same accession. Arachis duranensis and one accession of A. cardenasii were found to have identical protein patterns. The greatest dissimilarity was found between species of the section Extranervosae and species of the section Triseminalae. Those of section Erectoides showed much similarity with some species of section Arachis. Protein polymorphism was shown to distinguish the two subspecies of A. hypogaea (fastigiata and hypogaea) in 27 of 28 cases. The seed protein profile of A. monticola was a combination of seed protein profiles from the two A. hypogaea subspecies. The relatedness between the various species was calculated and those that had the greatest similarity with A. hypogaea were A. spegazzinii and A. batizocoi.     

Acetate inhibition on growth of recombinant E. coli and expression of fusion protein TGFaα-PE40

Summary Acetate was inhibitory to the growth of early induced E. coli cells and their expression of fusion protein, transforming growth factor--Pseudomonas exotoxin 40 (TGF-PE40), but the inhibitory level was strain dependent For E. coli JM109 (pTAC-TGF57-PE40), 2 g/L of added acetate (3 g/L of total acetate in the medium) decreased TGFa-PE40 production by 38.0%. Acetate was less inhibitory to E. coli RR1, and RR1 was not affected by adding 2 g/L of acetate. However, 5 g/L of added acetate (6.7 g/L of total acetate in the medium) decreased TGF-PE40 production by 21.2%. These results indicate that higher acetate concentration was associated with inhibition of TGF-PE40 expression of E. coli JM109 during late induction.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain.

To examine the role of the glycans of human immunodeficiency virus type 1 transmembrane glycoprotein gp41, conserved glycosylation sites within the env sequence (Asn-621, Asn-630, and Asn-642) were mutated to Gln. The mutated and control wild-type env genes were introduced into recombinant vaccinia virus and used to infect BHK-21 or CD4+ CEM cells. Mutated gp41 appeared as a 35-kDa band in a Western blot (immunoblot), and it comigrated with the deglycosylated form of wild-type gp41. Proteolytic cleavage of the recombinant wild-type and mutant forms of the gp160 envelope glycoprotein precursor was analyzed by pulse-chase experiments and enzyme-linked immunosorbent assay: gp160 synthesis was similar whether cells were infected with control or mutated env-expressing recombinant vaccinia virus, but about 10-fold less cleaved gp120 and gp41 was produced by the mutated construct than the control construct. The rates of gp120-gp41 cleavage at each of the two potential sites appeared to be comparable in the two constructs. By using a panel of antibodies specific for gp41 and gp120 epitopes, it was shown that the overall immunoreactivities of control and mutated gp41 proteins were similar but that reactivity to epitopes at the C and N termini of gp120, as present on gp160 produced by the mutated construct, was enhanced. This was no longer observed for cleaved gp120 in supernatants. Both gp120 proteins, from control and mutated env, were expressed on the cell surface under a cleaved form and could bind to membrane CD4, as determined by quantitative immunofluorescence assay. In contrast, and despite sufficient expression of env products at the cell membrane, gp41 produced by the mutated construct was unable to induce membrane fusion. Therefore, while contradictory results reported in the literature suggest that gp41 individual glycosylation sites are dispensable for the bioactivity and conformation of env products, it appears that such is not the case when the whole gp41 glycan cluster is removed.