Four-way ligation for construction of a mammalian cell-based full-length antibody display library

A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.     

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF I_m—are present in Drosophila nuclear extracts in a stable supercomplex.     

Synthesis and biological activity of dialkylphosphocholines

A series of dialkylphosphocholines were prepared and evaluated for their biological activity. The antiprotozoal activity was determined against Acanthamoeba lugdunensis. Compound 15 exhibited excellent trophocidal activity. None of the tested dialkylphosphocholines exhibited better fungicidal activity against Candida albicans than miltefosine. The antineoplastic activity was determined against HeLa. The most cytotoxic was compound 10, which was more active against tumor cells as against normal cells.     

The newly synthesized plant growth regulator <Emphasis Type="Italic">S</Emphasis>-methylmethionine salicylate may provide protection against high salinity in wheat

High salinity is one of the major environmental factors limiting the productivity of crop species worldwide. Improving the stress tolerance of cultivated plants and thus increasing crop yields in an environmentally friendly way is a crucial task in agriculture. In the present work the ability of a new derivative, S-methylmethionine-salicylate (MMS), to improve the salt tolerance of wheat plants was tested parallel with its related compounds salicylic acid and S-methylmethionine. The results show that while these compounds are harmful at relatively high concentration (0.5 mM), they may provide protection against high salinity at lower (0.1 mM) concentration. This was confirmed by gas exchange, chlorophyll content and chlorophyll-a fluorescence induction measurements. While osmotic adjustment probably plays a critical role in the improved salt tolerance, neither Na or K transport from the roots to the shoots nor proline synthesis are the main factors in the tolerance induced by the compounds tested. MMS, S-methylmethionine and Na-salicylate had different effects on flavonol biosynthesis. It was also shown that salt treatment had a substantial influence on the SA metabolism in wheat roots and leaves. Present results suggest that the investigated compounds can be used to improve salt tolerance in plants.     

Relations Between River Plant Richness in the Portuguese Floodplains and the Widespread Water Knotgrass <Emphasis Type="Italic">(Paspalum Paspalodes</Emphasis>)

We examined the variability of macroinvertebrate assemblage structure, species identities, and functional feeding group composition in relation to stream size, tributary position, and in-stream factors in a boreal watershed in Finland. Our study included three riffle sites in each of three stream sections in each of three stream size classes. Multi-response permutation procedure, indicator value method, and canonical correspondence analysis revealed clear differences in assemblage structure among the stream size classes, with a gradual increase of species richness as the stream size increased. Significant differences in assemblage structure were also found among the tributary river systems. The functional feeding group composition broadly followed the river continuum concept, i.e., headwaters were dominated by shredders, gatherers, or filterers, whereas scrapers increased in relative abundance with stream size. There was, however, considerable variation in the functional feeding group composition both among and within the headwater stream sections. Our findings refer to a strong influence of stream size on macroinvertebrate assemblages, but also factors prevailing at the scale of individual riffles should be considered in biodiversity conservation of lotic ecosystems.     

A comparative histological study between normal and fasciated shoots of <Emphasis Type="Italic">Prunus avium</Emphasis> generated <Emphasis Type="Italic">in vitro</Emphasis>

A combination of Murashige and Skoogs medium and N⁶–benzyladenine (BA) at various concentrations (0, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg l^–1) was supplied to shoot tips from root cuttings of a 50-year-old wild-cherry tree (Prunus avium). The concentration of BA in the growing medium was a determining factor with respect to the number of proliferated shoots per explant in vitro.Normal and fasciated shoots were generated when BA was present at 0.5, 0.75, 1.0 and 1.25 mg l^–1 in the medium and the mean numbers of normal shoots per explant were 3.63, 5.37, 8.93 and 7.30 respectively, and those of the fasciated shoots per explant were 0.03, 0.1, 0.47 and 0.4 respectively. Anatomical analysis by confocal microscopy of sections of paraffin-embedded specimens revealed that the cell structure and organization of the cortex and vascular cylinder in the fasciated shoots was similar to that in normal shoots. However, the cross-sectional area of stem of the fasciations was apparently greater than that of the normal shoots. In particular, the volume of vascular tissues, of pith and of some individual parenchyma cells in the cortex and pith was apparently greater in fasciated shoots than in normal shoots. Increases in cytokinesis and morphogenetic activity, such as the development of callus-like regions and the formation of adventitious shoots, were observed in the cortex and pith throughout the fasciations. The fasciated shoots had numerous buds and initiating new shoots at their apices while normal shoots had a single dominant axial bud.