Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.     

SIDECAR POLLEN suggests a plant-specific regulatory network underlying asymmetric microspore division in Arabidopsis

Asymmetric cell division is a universal strategy to generate diverse cell types necessary for patterning and proliferation of all eukaryotes. The development of haploid male gametophytes (pollen grains) in flowering plants is a remarkable example in which division asymmetry governs the functional specialization and germline differentiation essential for double fertilization. The male gametophyte is patterned via two mitotic divisions resulting in three highly differentiated daughter cells at maturity, a vegetative cell and two sperm cells. The first asymmetric division segregates a unique male germ cell from an undetermined haploid microspore and is executed in an elaborate sequence of cellular events. However the molecular mechanisms governing the division asymmetry in microspores are poorly understood. Recently we studied the phenotype of sidecar pollen (scp) mutants in detail, and demonstrated a requirement of SCP for both the correct timing and orientation of microspore division. SCP is a microspore-specific member of the LOB/AS2 domain family (LBD27/ASL29) showing that a plant-specific regulator plays a key role in oriented division of polarized microspores. Identification of SCP will serve as a new platform to further explore the largely unknown molecular networks regulating division asymmetry in microspores that establishes the male germline in flowering plants.Key words: sidecar pollen, microspore division, division asymmetry, male gametophyte development, male germline, LBD/ASL family proteinUnlike animals, flowering plants do not set aside a distinct germline from an early stage of the life cycle. Instead the angiosperm germline or germ cells are only segregated in the male and female gametophytes by a limited number of post-meiotic mitoses.¹ However, in common with their metazoan cousins, angiosperms utilize division asymmetry for cellular patterning and differentiation of their germlines. Through the unique patterning of a ‘cell-within-a-cell’ structure with three highly differentiated cells, the male gametophyte (pollen grains) serves its biological role to deliver two sessile male gametes to the female gametophyte. Two sequential but different modes of mitotic divisions pattern the male gametophyte (Fig. 1).² The first division (of the microspore) is asymmetric giving rise to two completely different daughter cells, a larger vegetative cell that will form the pollen tube and a smaller germ cell that is engulfed within the vegetative cell cytoplasm. The second division (of the germ cell) usually appears symmetric^† and produces a pair of linked sperm cells. Microspores artificially induced to undergo symmetric division using microtubule inhibitors lack the germ cell and fail to form the typical three-celled structure showing that asymmetry in microspore division is critical for patterning of the male gametophyte.⁴Open in a separate windowFigure 1Male gametophyte development in Arabidopsis (upper part) and mutations that block germ cell formation (lower part). (Upper part) Male gametophyte development involves two rounds of mitotic division. Prior to the first division the centrally positioned microspore nucleus migrates towards the radial wall (the future germ cell pole marked with an asterisk). At this eccentric site the polarized microspores undergo oriented mitosis and cytokinesis giving rise to highly unequal daughter cells, a vegetative cell and a germ cell of which the later produces a pair of sperm cells by symmetric division. (Lower part) Mutants that fail to establish a distinct germ cell arising from specific defects are illustrated. Arrows in red indicate the developmental origin of the phenotypic defects in mutants. Note that two daughter nuclei in the mutants are in grey to show that their cell fates have not yet been thoroughly investigated. n, nucleus; Vn, vegetative nucleus; Gn, generative nucleus; Gc, generative (or germ) cell; Sc, sperm cell; WT, wild type; gem1, gemini pollen1; scp, sidecar pollen; tio, two-in-one; hik/tes, hinkel/tetraspore 12a/12b, kinesin-12a/kinesin-12b.     

A monoclonal antibody specific for Candida albicans Als4 demonstrates overlapping localization of Als family proteins on the fungal cell surface and highlights differences between Als localization in vitro and in vivo

The Candida albicans agglutinin-like sequence (ALS) family encodes large cell surface glycoproteins that function in adhesion of the fungus to host and abiotic surfaces. Monoclonal antibodies (mAbs) specific for each Als protein were developed to study Als localization on the C. albicans surface. An anti-Als4 mAb demonstrated that Als4 covers the surface of yeast cells, with a greater abundance of Als4 on cells grown at 30 °C compared to 37 °C. On germ tubes, Als4 is localized in a restricted area proximal to the mother yeast. Immunolabeling with several anti-Als mAbs showed overlapping localization of Als1 and Als4 on yeast cells and Als1, Als3 and Als4 on germ tubes. Overlapping localization of Als proteins was also observed on yeast and hyphae recovered from mouse models of disseminated and oral candidiasis. Differences between Als localization in vivo and in vitro suggested changes in regulation of Als production in the host compared to the culture flask. Characterization with the anti-Als mAbs reveals the simultaneous presence and differences in relative abundance of Als proteins, creating an accurate image of Als representation and localization that can be used to guide conclusions regarding individual and collective Als protein function.     

Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate

Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. (c) 1993 John Wiley & Sons, Inc.     

三峡水库社区渔业浅析

文章考察了国内外社区渔业的发展概况, 并基于三峡水库的实际情况开展了渔业社区的构建和运行试验, 结果显示, 社区渔业在三峡库湾实现了生态效益、经济效益和社会效益, 具有综合效应, 将是养护三峡渔业资源、开展渔业生产的一种重要组织方式, 可确保三峡渔业的可持续发展。同时, 通过实践还确认了三峡水库实施社区渔业存在现实的困难, 渔业社区的构建和运营在准入制度、管理体制等诸多方面存在问题,三峡水库发展社区渔业需要政策引导和资金扶持, 同时需要严格的管理制度确保良好的从业环境。       

The ATP-dependent RNA helicase,DDX42 interacts with paxillin and regulates apoptosis and polarization of Ba/F3 cells

Paxillin is a focal adhesion adaptor protein, heavily phosphorylated at multiple tyrosine residues, as well as at serine 273 (S273), and is known to be critical for cytoskeleton rearrangement and cell migration. We previously found that paxillin plays a regulatory role in IL-3-dependent survival of Ba/F3 cells, a mouse pro-B cell line. In this study, by using overexpressed His6 tagged-paxillin as a bait, we found that DDX42, a DEAD-box RNA helicase, interacted with paxillin, inhibited apoptosis, and promoted polarization of Ba/F3 cells. His6 tagged-paxillin was stably overexpressed in Ba/F3 cells, pulled-down from cell lysates with Ni⁺-NTA beads, and analyzed by one-dimensional SDS-PAGE followed by LC–MS. We found that DDX42 co-precipitated with paxillin, as demonstrated by western blotting analysis of His6 tagged-paxillin precipitates with anti-DDX42 antibodies and His6 tagged-DDX42 precipitates with anti-paxillin antibodies. In addition, we observed a preferential interaction of DDX42 with the paxillin mutant, S273A, compared to the S273D mutant. Furthermore, DDX42 overexpression in Ba/F3 cells delayed the apoptosis induced by IL-3 deprivation and promoted restoration of the elongated shape in Ba/F3 cells induced by IL-3 re-supply after a 6?h-deprivation. These results suggested that DDX42 interacts with paxillin and participates in IL-3-dependent cell survival, as well as in the cytoskeletal rearrangements underlying polarization of Ba/F3 cells.